Compounds and methods for treating inflammatory diseases

ABSTRACT

A monoclonal secretory IgA antibody, which binds to and neutralizes a human proinflammatory cytokine or which binds to and blocks a human proinflammatory cytokine receptor. The secretory IgA antibody is useful in treating a variety of inflammatory diseases in humans.

This application claims the benefit of priority under 35 U.S.C. §119(e)from U.S. provisional patent application Ser. No. 61/576,727, filed Dec.16, 2011, and Ser. No. 61/576,922, filed Dec. 16, 2011; the entirecontents of each provisional application being incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodiesthat bind to and neutralize one or more human proinflammatory cytokines,or bind to and block a human receptor of a human proinflammatorycytokine. The antibodies are useful in treating inflammatory diseases inhumans.

BACKGROUND OF THE INVENTION

Inflammation represents a key event of many diseases, such as psoriasis,inflammatory bowel diseases, rheumatoid arthritis, asthma, multiplesclerosis, atherosclerosis, cystic fibrosis, and sepsis. Inflammatorycells, such as neutrophils, eosinophils, basophils, mast cells,macrophages, endothelial cells, and platelets, respond to inflammatorystimuli and foreign substances by producing bioactive mediators. Thesemediators act as autocrines and paracrines by interacting with many celltypes to promote the inflammatory response. There are many mediatorsthat can promote inflammation, such as cytokines and their receptors,adhesion molecules and their receptors, antigens involved in lymphocyteactivation, and IgE and its receptors.

Cytokines, for example, are soluble proteins that allow forcommunication between cells and the external environment. The termcytokines includes a wide range of proteins, such as lymphokines,monokines, interleukins, colony stimulating factors, interferons, tumornecrosis factors, and chemokines. Cytokines serve many functions,including controlling cell growth, migration, development, anddifferentiation, and mediating and regulating immunity, inflammation,and hematopoiesis. Even within a given function, cytokines can havediverse roles. For example, in the context of mediating and regulatinginflammation, some cytokines inhibit the inflammatory response(anti-inflammatory cytokines), others promote the inflammatory response(pro-inflammatory cytokines). And certain cytokines fall into bothcategories, i.e., can inhibit or promote inflammation, depending on thesituation. The targeting of proinflammatory cytokines to suppress theirnatural function, such as with antibodies, is a well-establishedstrategy for treating various inflammatory diseases.

Many inflammatory diseases are treated by targeting proinflammatorycytokines with antibodies. Most (if not all) of the anti-proinflammatorycytokine antibodies currently on the market, and those currently inclinical trials, are of the IgG class. See, for example, Nature Reviews,vol. 10, pp. 301-316 (2010); Nature Medicine, vol. 18, pp. 736-749(2012); Nature Biotechnology, vol. 30, pp. 475-477 (2012);Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, vol. 8,pp. 51-71 (2009); F1000.com/Reports/Biology/content/1/70, F1000 BiologyReports, 1:70 (2009); mAbs 4:1, pp. 1-3 (2012); mAbs 3:1, pp. 76-99(2011); clinicaltrials.gov (generally), and clinicaltrialsregister.eu/(generally). These IgG antibodies are administered systemically and thusare often associated with unwanted side effects, which can include oneor more of, for example, infusion reactions and immunogenicity,hypersensitivity reactions, immunosuppression and infections, heartproblems, liver problems, and others. Additionally the suppression ofthe target cytokines at non-diseased parts of the body can lead tounwanted effects.

In an attempt to reduce side effects associated with systemic treatmentand to eliminate the inconvenience and expense of infusions, an articleproposed an oral anti-TNF therapy that could be useful in treatingCrohn's disease. Worledge et al. “Oral Administration of Avian TumorNecrosis Factor Antibodies Effectively Treats Experimental Colitis inRats.” Digestive Diseases and Sciences 45(12); 2298-2305 (December2000). This article describes immunizing hens with recombinant human TNFand an adjuvant, fractionating polyclonal yolk antibody (IgY, which inchickens is the functional equivalent to IgG), and administering theunformulated polyclonal IgY (diluted in a carbonate buffer to minimizeIgY acid hydrolysis in the stomach) to rats in an experimental rodentmodel of colitis. The rats were treated with 600 mg/kg/day of thepolyclonal IgY. The uses of animal antibodies and polyclonal antibodies,however, are undesirable.

In a similar attempt to avoid adverse events associated with systemicadministration, another group, Avaxia Biologics Inc., describes atopical (e.g., oral or rectal) animal-derived polyclonal anti-TNFcomposition that could be useful in treating inflammation of thedigestive tract, such as inflammatory bowel disease. WO2011047328. Theapplication generally states that preferably the polyclonal antibodycomposition is prepared by immunizing an animal with a target antigen,and the preferably the polyclonal antibody composition is derived frommilk or colostrum with bovine colostrums being preferred (e.g., p. 14).The application also generally states that the animal derived polyclonalantibodies could be specific for (among other targets) otherinflammatory cytokines (e.g., pp. 6-7). This application describesworking examples in which cows were immunized with murine TNF and thecolostrum was collected post-parturition to generate bovine polyclonalanti-TNF antibodies (designated as AVX-470). The uses of animal-derivedantibodies and polyclonal antibodies, however, are undesirable.

IgA molecular forms have been proposed as treatments for variousdiseases, most notably as treatments for pollen allergies, as treatmentsagainst pathogens, and as treatments for cancer.

For example, one article describes anti-AmbαI (a ragweed pollen antigen)humanized monomeric IgA and dimeric IgA antibodies made in murine cells(NSO and Sp2/0 cells). The dimeric IgA contains a mouse J-chain. Thearticle proposes that the antibodies may be applied to a mucosal surfaceor the lower airway to inhibit entry of allergenic molecules across themucosal epithelium and therefore to prevent the development of allergicresponse. Sun et al. “Human IgA Monoclonal Antibodies Specific for aMajor Ragweed Pollen Antigen.” Nature Biotechnology 13, 779-786 (1995).

Several other articles propose the use of IgA antibodies as a defenseagainst pathogens.

Two articles proposed the use of an anti-streptococcal antigen I/IIsecretory IgA-G hybrid antibody. Ma et al. “Generation and Assembly ofSecretory Antibodies in Plants.” Science 268(5211), 716-719 (May 1995);Ma et al. “Characterization of a Recombinant Plant Monoclonal SecretoryAntibody and Preventive Immunotherapy in Humans.” Nature Medicine 4(5);601-606 (May 1998). The hybrid antibody contains murine monoclonal kappalight chain, hybrid Ig A-G heavy chain, murine J-Chain, and rabbitsecretory component. The antibody was made by successive sexual crossingbetween four transgenic N. tabacum plants and filial recombinants toform plant cells that expressed all four protein chains simultaneously.The parent antibody (the source of the antigen binding regions, isidentified as the IgG antibody Guy's 13. The group proposes thatalthough sIgA may provide an advantage over IgG in the mucosalenvironment, such is not always the case (1998 Ma at p. 604, rightcolumn).

A related article identifies the anti-streptococcal antigen I/IIsecretory IgA-G hybrid antibody, which was derived from Guy's 13 IgA, asCaroRx. Wycoff. “Secretory IgA Antibodies from Plants.” CurrentPharmaceutical Design 10(00); 1-9 (2004). Planet Biotechnology Inc. Thisrelated article states that the CaroRx antibody was designed to blockadherence to teeth of the bacteria that causes cavities. Apparently, theCaroRx antibody was difficult to purify; the affinity of Protein A forthe murine Ig domain was too low and protein G was necessary forsufficient affinity chromatography. Furthermore, the article states thatseveral other chromatographic media had shown little potential aspurification steps for the hybrid sIgA-G from tobacco leaf extracts. Thearticle also indicates that the authors were unable to control forhuman-like glycosylation in tobacco, but that such was not a problembecause people are exposed to plant glycans every day in food withoutill effect.

WO9949024, which lists Wycoff as an inventor, Planet Biotechnology Inc.as the applicant, describes the use of the variable regions of Guy's 13to make a secretory antibody from tobacco. The application contains onlytwo examples—the first a working example and the second a propheticexample. Working Example 1 describes the transient production of ananti-S. mutans SA I/III (variable region from Guy's 13) in tobacco. Thetobacco plant was transformed using particle bombardment of tobacco leafdisks. Transgenic plants were then screened by Western blot “to identifyindividual transformants expressing assembled human sIgA” (p. 25).Prophetic Example 2 states that in a transformation system for Lemnagibba (a monocot), bombardment of surface-sterilized leaf tissue withDNA-coated particles “is much the same as with” tobacco (a dicot). Theprophetic example also stops at screening by immunoblot analysis forantibody chains and assembled sIgA, and states that the inventors“expect to find fully assembled sIgA.”

Another article proposed the use of an anti-RSV glycoprotein F IgAantibodies (mIgA, dIgA, and sIgA). Berdoz et al. “In vitro Comparison ofthe Antigen-Binding and Stability Properties of the Various MolecularForms of IgA antibodies Assembled and Produced in CHO Cells.” Proc.Natl. Acad. Sci. USA 96; 3029-3034 (March 1999). The sIgA antibody wasmade in CHO cells sequentially transfected with chimeric heavy and lightchains, human J-Chain, and human secretory component, respectively.Single clones were generated to express the mIgA (clone 22), the dIgA(clone F), and the sIgA (clone 6) (p. 3031).

Still other articles proposed, for example: (1) anti-HSV mIgA made inmaize (Karnoup et al. Glycobiology 15(10); 965-981 (May 2005)) (whichstates that at that time there had been little success in theapplication of IgA class antibodies to therapeutic use because of thedifficulty in producing the dimeric form in mammalian cells at economiclevels); (2) anti-C. difficile toxin A chimeric mouse-human monomericand dimeric IgA made in CHO cells (Stubbe et al. Journal of Immunology164; 1952-1960 (2000)); (3) anti-N. meningitidis chimeric IgA antibodieswere produced in BHK cells cotransfected with human J-Chain and/or humansecretory component (Vidarsson et al., Journal of Immunology 166;6250-6256 (2001)); (4) anti-Pseudomonas aeruginosa O6 lipopolysaccharidechimeric mouse/human mIgA1 made in CHO cells (Preston et al. Infectionand Immunity 66(9); 4137-4142 (September 1998)); (5) anti-PlasmodiummIgA made in CHO cells (Pleass et al. Blood 102(13); 4424-4429 (December2003)) (which states that unlike their parental mouse IgG antibodies,the mIgA antibodies failed to protect against parasitic challenge invivo); and (5) anti-Helicobacter pylori urease subunit A sIgA and dIgA(Berdoz et al. Molecular Immunology 41(10); 1013-1022 (August 2004)).

For a review article discussing passive and active protection againstpathogens at mucosal surfaces, see Corthésy. “Recombinant ImmunoglobulinA: Powerful Tools for Fundamental and Applied Research.” Trends inBiotechnology 20(2); 65-71 (February 2002).

Still other articles propose the use of IgA antibodies as a treatmentfor cancer.

For example, one article describes a Phase Ia trial of a muringanti-transferrin receptor IgA antibody (Brooks et al. “Phase Ia Trial ofMurine Immunoglobulin A Antitransferrin Receptor Antibody 42/6.”Clinical Cancer Research 1(11); 1259-1265 (November 1995)). Anotherarticle describes a human anti-Ep-CAM mIgA made in BHK (baby hamsterkidney) cells (Huls et al. “Antitumor Immune Effector MechanismsRecruited by Phase Display-Derived Fully Human IgG1 and IgA1 MonoclonalAntibodies.” Cancer Research 59; 5778-5784 (November 1999)). Stillanother article describes an anti-HLA Class II chimeric mIgA antibodymade in BHK cells (Dechant et al. “Chimeric IgA Antibodies Against HLAClass II Effectively Trigger Lymphoma Cell Killing.” Blood 100(13);4574-4580 (December 2002)). Yet other articles describe anti-EGFR mIgAor dIgA antibodies made in CHO, including Dechant et al. “EffectorMechanisms of Recombinant IgA Antibodies Against Epidermal Growth FactorReceptor.” Journal of Immunology 179; 2936-2943 (2007), Beyer et al.“Serum-Free Production and Purification of Chimeric IgA Antibodies.”Journal of Immunology 346; 26-37 (2009) (stating that as of 2009, IgAantibodies have not been commercially explored for problems includinglack of production and purification methods), and Lohse et al.“Recombinant Dimeric IgA Antibodies Against the Epidermal Growth FactorReceptor Mediate Effective Tumor Cell Killing” Journal of Immunology186; 3770-3778 (February 2011).

For a review article on anti-cancer IgA antibodies, see Dechant et al.“IgA antibodies for Cancer Therapy.”Critical Reviews inOncology/Hematology 39; 69-77 (2001); states that compared withinfectious diseases, the role of IgA in cancer immunotherapy is evenless investigated).

It is desirable to have alternative antibody treatments for inflammatorydiseases that preferably avoid the disadvantages of current systemic andpreviously-proposed topical treatments of inflammation.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodiesthat bind to and neutralize a human proinflammatory cytokine, or bind toand block a human receptor of a human proinflammatory cytokine, and theuse of such secretory IgA antibodies in treating inflammatory diseasesin humans.

An aspect of the present invention relates to a monoclonal secretory IgAantibody that binds to and neutralizes a human proinflammatory cytokine.

In embodiments, the antibody can be, for example a chimeric antibody, ahumanized antibody, or a human antibody. The antibody can contain ahuman secretory chain and a human J-chain. The antibody can be a humansecretory IgA1 antibody. The human proinflammatory cytokine can beselected from, for example, TNFα (soluble), interferon gamma, interferonalpha, GM-CSF, CXCL10/IP-10, IL-1β, IL-1α, IL-4, IL-5, IL-6, IL-12;IL-13, IL-17A, IL-18, IL-20, IL-22, and IL-23. The antibody can containCDR sequences that are identical to the CDR sequences of an antibodyselected from adalimumab, infliximab, golimumab, certolizumab pegol,ozoralizumab, sifalimumab, canakinumab, gevokizumab, pascolizumab,reslizumab, mepolizumab, sirukumab, olokizumab, ustekinumab,briakinumab, tralokinumab, anrukinzumab, lebrikizumab, secukinumab,ixekizumab, and fezakinumab.

Another aspect of the present invention relates to a compositioncontaining a plurality of the secretory IgA antibodies. In embodiments,substantially all N-glycans in the plurality of antibodies lack fucoseand xylose residues. In embodiments, the plurality of antibodiescontains at least about 30% G0 glycans (preferably G0 glycans lackingFuc and Xyl residues) relative to the total amount of N-glycans in thepopulation. In embodiments, the a plurality of antibodies ac contains atleast about 25% high-mannose glycans (e.g., Man5, Man6, Man7, Man8,and/or Man9 glycans) relative to the total amount of N-glycans in thepopulation. In embodiments, the G0 glycans (preferably G0 glycanslacking Fuc and Xyl residues) and high-mannose glycans (e.g., Man5,Man6, Man7, Man8, and/or Man9 glycans) together are the majority ofglycans present in said plurality of antibodies, such as at least 70% ofthe total amount of N-glycans in said plurality of antibodies.

Another aspect of the present invention relates to pharmaceuticalcompositions containing the secretory IgA antibodies, which can beadapted for oral administration and can be used to treat an inflammatorydisease in a human.

Another aspect of the present invention relates to methods for treatingan inflammatory disease in a human, which includes administering ananti-inflammatory effective amount of the secretory IgA antibodies (orcompositions) to a human in need thereof, preferably orallyadministering the antibodies (compositions). The inflammatory diseasecan be selected from rheumatoid arthritis, inflammatory bowel disease(including Crohn's disease and ulcerative colitis), psoriasis, psoriaticarthritis, ankylosing spondylitis, juvenile idiopathic arthritis,uveitis, asthma, Alzheimer's disease, multiple sclerosis, type IIdiabetes, systemic sclerosis, lupus nephritis, and allergic rhinitis.

Another aspect of the present invention relates to a monoclonalsecretory IgA antibody that binds to and blocks a human proinflammatorycytokine receptor selected from IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, andIL-17R.

Brief Description of the Sequences

SEQ ID NO:1 provides the amino acid sequence of the soluble TNF-αreceptor of etanercept (extracellular part of tumor necrosis factorreceptor 2: amino acids 23-257 of UniProtKB/Swiss-Prot database entryP20333 (TNR1B_HUMAN)).

SEQ ID NO:2 provides the amino acid sequence of the heavy chain variableregion of infliximab (cA2).

SEQ ID NO:3 provides the amino acid sequence of the light chain variableregion of infliximab (cA2).

SEQ ID NO:4 provides the amino acid sequence of the heavy chain variableregion of adalimumab (D2E7).

SEQ ID NO:5 provides the amino acid sequence of the light chain variableregion of adalimumab (D2E7).

SEQ ID NO:6 provides the amino acid sequence of the heavy chain variableregion of golimumab.

SEQ ID NO:7 provides the amino acid sequence of the light chain variableregion of golimumab.

SEQ ID NO:8 provides the amino acid sequence of the heavy chain variableregion of certolizumab pegol.

SEQ ID NO:9 provides the amino acid sequence of the light chain variableregion of certolizumab pegol.

SEQ ID NO:10 provides the amino acid sequence of a human IgA α-1 heavychain constant region (Cα1-Cα2-Cα3) (UniProtKB/Swiss-Prot database entryP01876 (IGHA1_HUMAN)).

SEQ ID NO:11 provides the amino acid sequence of a human IgA α-2m(1)-allotype heavy chain constant region (Cα1-Cα2-Cα3)(UniProtKB/Swiss-Prot database entry P01877 (IGHA2_HUMAN)).

SEQ ID NO:12 provides the amino acid sequence of a human IgA α-2m(2)-allotype heavy chain constant region (Cα1-Cα2-Cα3).(UniProtKB/Swiss-Prot database entry P01877 (IGHA2_HUMAN) with indicatedmodifications for allotype 2 variant).

SEQ ID NO:13 provides the amino acid sequence of a human IgA α-2(n)-allotype.

SEQ ID NO:14 provides the amino acid sequence of a human κ light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry P01834(IGKC_HUMAN)).

SEQ ID NO:15 provides the amino acid sequence of a human λ1 light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry P0CG04(LAC1_HUMAN)).

SEQ ID NO:16 provides the amino acid sequence of a human λ2 light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry P0CG05(LAC2_HUMAN)).

SEQ ID NO:17 provides the amino acid sequence of a human λ3 light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry P0CG06(LAC3_HUMAN)).

SEQ ID NO:18 provides the amino acid sequence of a human λ6 light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry P0CF74(LAC6_HUMAN)).

SEQ ID NO:19 provides the amino acid sequence of a human λ7 light chainconstant region (C_(L)) (UniProtKB/Swiss-Prot database entry A0M8Q6(LAC7_HUMAN)).

SEQ ID NO:20 provides the amino acid sequence of a human J-chain (aminoacids 23-159 UniProtKB/Swiss-Prot database entry P01591).

SEQ ID NO:21 provides the amino acid sequence of a human secretorycomponent (amino acids 19-603 of UniProtKB/Swiss-Prot database entryP01833 (PIGR_HUMAN), RCSB Protein Data Bank structure 2OCW).

SEQ ID NO:22 provides the amino acid sequence of a natural signalpeptide (secretion signal) for a human J-chain (amino acids 1-22UniProtKB/Swiss-Prot database entry P01591).

SEQ ID NO:23 provides the amino acid sequence for a signal peptide(heavy chain secretion signal).

SEQ ID NO:24 provides the amino acid sequence for a signal peptide(light chain secretion signal).

SEQ ID NO:25 provides the amino acid sequence for a signal peptide(SC-chain secretion signal).

SEQ ID NO:26 provides the amino acid sequence for a rice α-amylasesignal peptide (secretion signal).

SEQ ID NO:27 provides the DNA sequence for the infliximab heavy chainIgA2m(n) optimized for maize.

SEQ ID NO:28 provides the DNA sequence for the infliximab heavy chainIgA1 and light chain κ optimized for HEK.

SEQ ID NO:29 provides the DNA sequence for the infliximab light chainoptimized for maize.

SEQ ID NO:30 provides the DNA sequence for SC- and J-chains optimizedfor HEK.

SEQ ID NO:31 provides the DNA sequence for the adalimumab heavy chainIgA1 optimized for Lemna.

SEQ ID NO:32 provides the DNA sequence for the adalimumab light chain κoptimized for Lemna.

SEQ ID NO:33 provides the DNA sequence for a J-chain optimized formaize.

SEQ ID NO:34 provides the DNA sequence for a J-chain optimized forLemna.

SEQ ID NO:35 provides the DNA sequence for an SC-chain optimized formaize.

SEQ ID NO:36 provides the DNA sequence for an SC-chain optimized forLemna.

SEQ ID NO:37 provides the amino acid sequence of the heavy chainvariable region of ustekinumab (CTNO-1275).

SEQ ID NO:38 provides the amino acid sequence of the light chainvariable region of ustekinumab (CTNO-1275).

SEQ ID NO:39 provides the amino acid sequence of the heavy chainvariable region of briakinumab (J-695, ABT-874).

SEQ ID NO:40 provides the amino acid sequence of the light chainvariable region of briakinumab (J-695, ABT-874).

SEQ ID NO:41 provides a complete lemna-optimized UKB-SA1 heavy chainIgA1 DNA (including DNA encoding signal peptide SEQ ID NO:26).

SEQ ID NO:42 provides a complete lemna-optimized UKB-SA1 light chain DNA(including DNA encoding signal peptide SEQ ID NO:26).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the constant regions of humanIgA1 (SEQ ID NO:10), IgA2 m(1) (SEQ ID NO:11), IgA2 m(2) (SEQ ID NO:12),and IgA2(n) (SEQ ID NO:13) antibody a heavy chains;

FIGS. 2A through 21 show the amino acid sequence of various TNF-αbinding regions. FIG. 2A shows the amino acid sequence of the solubleTNF-α receptor of etanercept (SEQ ID NO:1); FIG. 2B shows the amino acidsequence of the heavy chain variable region of infliximab (cA2) (SEQ IDNO:2); FIG. 2C shows the amino acid sequence of the light chain variableregion of infliximab (cA2) (SEQ ID NO:3); FIG. 2D shows the amino acidsequence of the heavy chain variable region of adalimumab (D2E7) (SEQ IDNO:4); FIG. 2E shows the amino acid sequence of the light chain variableregion of adalimumab (D2E7) (SEQ ID NO:5); FIG. 2F shows the amino acidsequence of the heavy chain variable region of golimumab (SEQ ID NO:6);FIG. 2G shows the amino acid sequence of the light chain variable regionof golimumab (SEQ ID NO:7); FIG. 2H shows the amino acid sequence of theheavy chain variable region of certolizumab pegol (SEQ ID NO: 8); FIG.2I shows the amino acid sequence of the light chain variable region ofcertolizumab pegol (SEQ ID NO:9).

FIGS. 3A and 3B show the amino acid sequences of various human antibodylight chain subtypes and allotypes. FIG. 3A shows the amino acidsequence of a human κ light chain constant region (C_(L))(UniProtKB/Swiss-Prot P01834) (SEQ ID NO:14); FIG. 3B shows the aminoacid sequences of a human λ light chain constant region (C_(L))allotypes (UniProtKB/Swiss-Prot P0CG04, P0CG05, P0CG06, P0CG74, andA0M8Q6; SEQ ID NOS:15 to 19).

FIG. 4 shows the amino acid sequence of a human J-chain (a.a. 23-159 ofUniProtKB/Swiss-Prot entry P01591, SEQ ID NO:20).

FIG. 5 shows the amino acid sequence of a human secretory component(a.a. 19-603 of UniProtKB/Swiss-Prot database entry P01833 [PIGR_HUMAN],SEQ ID NO:21).

FIG. 6 shows the structure of the SIgA vector constructs of Example 1.

FIG. 7 shows reducing and non reducing gels of an anti-TNFα SIgA of theinvention, having infliximab variable regions and being produced inmammalian HEK-293F cells. The label “A” shows non-reducing SDS-PAGEanalysis demonstrating expression of complete SIgA while the label “B”shows reducing SDS-PAGE analysis.

FIG. 8 shows reducing and non reducing gels of an anti-TNFα SIgA of theinvention, having adalimumab variable regions and being produced inLemna. The label “A” shows non-reducing SDS-PAGE analysis demonstratingexpression of complete SIgA while the label “B” shows reducing SDS-PAGEanalysis.

FIG. 9 shows binding curves of antibodies to TNFα. The closed circlerepresents a SIgA of the invention having infliximab variable regions,square represents infliximab, and diamond represents colostral secretoryIgA.

FIG. 10 shows the degradation of an anti-TNFα SIgA according to theinvention having infliximab variable regions and being produced inmammalian cells, and an anti-TNFα SIgA according to the invention havingadalimumab variable regions and being produced in Lemna, compared tocolostral SIgA in simulated intestinal fluid (SIF).

FIG. 11 shows TNFα-induced disturbance of Caco-2 monolayer integrity asmeasured by transepithelial electrical resistance (TER).

FIG. 12 shows the restoration of TNFα-induced disturbance of Caco-2monolayer integrity as measured by TER.

FIG. 13 represents the colitis scoring of DSS-induced inflammatory boweldisease in C57BL/6 mice with and without treatment.

FIG. 14 is a representative depiction of mini-endoscopic pictures of thecolitis score at day t=15 for a mouse from each test group.

FIG. 15 shows the effects of subcutaneous application of etanercept andof oral application of ADB-SA1g on gripping strength of a transgenicTNFα-overexpressing mice that spontaneously develop arthritis(TNF^(ΔARE/+)-mice).

FIG. 16 shows the effects of subcutaneous application of etancercept andof oral application of ADB-SA1g on arthritic score of hind paws oftransgenic TNFα-overexpressing mice which spontaneously developarthritis (TNF^(ΔARE/+)-mice).

FIGS. 17A through 17D shows amino acid sequences of various p40 (the p40subunit of IL12 and IL23) binding regions. FIG. 17A shows the amino acidsequence of the heavy chain variable region of the antibody ustekinumab(CTNO-1275) (SEQ ID NO:37); FIG. 17B shows the amino acid sequence ofthe light chain variable region of the antibody ustekinumab (CTNO-1275)(SEQ ID NO:38); FIG. 17C shows the amino acid sequence of the heavychain variable region of the antibody briakinumab (J-695, ABT-874) (SEQID NO:39); FIG. 17D shows the amino acid sequence of the light chainvariable region of the antibody briakinumab (J-695, ABT-874) (SEQ IDNO:40).

FIGS. 18A through 18C show the structure of vector constructs SynA01(FIG. 18A), SynA02 (FIG. 18B) and SynA03 (FIG. 18C) used for expressionof anti-IL12/23 SIgA in Lemna in Example 1.

FIG. 19 shows reducing and non reducing gels of an anti-IL-12/23 SIgAaccording to the invention, having ustekinumab variable regions andbeing produced in Lemna. The A gel shows non-reducing SDS-PAGE analysisdemonstrating expression of complete SIgA. The B gel shows reducingSDS-PAGE analysis.

FIG. 20 shows the degradation of an anti-IL-12/23 SIgA according to theinvention having ustekinumab variable regions and being expressed inLemna compared to ustekinumab (IgG1) and to colostral secretory IgA insimulated intestinal fluid (SIF). Gel A compares the SynA01-WT antibodyof the invention (UKB-SA1) and gel B compares the SynA01-GO antibody ofthe invention (UKB-SA1g0).

FIGS. 21A and 21B show the results of inhibition of IL-12/23 (p40)production by LPS-stimulated Dendritic Cells from Examples 15A and 15B,respectively. In FIG. 21A, the open circle is SynA01 (UKB-SA1); theclosed circle is SynA02 (UKB-SA1g0); the diamond is colostral secretoryIgA; and the square is ustekinumab.

FIGS. 21C and 21D show the results of inhibition of production of IFNγby co-cultured dendritic cells and T-Cells from Examples 15C and 15D,respectively.

FIG. 22 shows the serum concentrations of SynA02 (UKB-SA1g0) after oral(circle) and intravenous (square) administration as described in Example16.

FIG. 23 shows in vivo imaging of fluorescent labeled SynA01 (UKB-SA1)and ustekinumab antibodies in the thoracic and abdominal region of miceas described in Example 17.

FIG. 24 shows immunohistochemistry on cryo-sections of mice distal colonafter administration of SynA01 (UKB-SA1) and ustekinumab antibodies asdescribed in Example 18.

FIGS. 25A and 25B show the efficacy of anti-IL12/23 SIgA in in vivoanimal models of IBD as described in Example 19.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodiesthat bind to and neutralize a human proinflammatory cytokine, or bind toand block a human receptor of a human proinflammatory cytokine, and theuse of such secretory IgA antibodies in treating inflammatory diseasesin humans.

As is well known, the basic structural unit of an antibody consists oftwo heavy chain proteins (heavy chains) and two light chain proteins(light chains), which are bound together by non-covalent and covalent(e.g., disulfide bonds) interactions into a single unit. The heavy andlight chains have N-terminal variable regions and C-terminal constantregions. The variable regions of the light and heavy chains togetherform an “antigen binding region.” Because the antibody has two heavy andlight chains, the antibody has two antigen binding regions.

Antibodies are classified based on the heavy chain constant region,e.g., classified as IgG, IgA, IgM, IgE, IgD, etc. The light chainconstant region is not used for classification. In humans, for example,all classes use one of two types of light chain constant regions, namelythe Cκ (kappa) or Cλ (lambda) type. The amino acid sequences of humankappa (SEQ ID NO:14) and several lambda light chain constant regions(SEQ ID NOS:15-19) are provided in FIGS. 3A and 3B, respectively. Innature, the heavy chain constant regions of the various classes areproduced by different genes: the IgA class heavy chains are uniquelyencoded-for by α genes, the IgG class heavy chains by γ genes, and soforth. The heavy chain constant regions also impart the various classeswith differences in their physio-chemical properties, their isotypicantigentic determinants, and/or in their biological function. Lefranc etal., The Immunoglobulin FactsBook, Academic Press 2001, Chapter 2, (ISBN0-12-441351-X). The constant region of an IgA heavy chain (Ca) typicallyhas three domains that are referred to as Cα1, Cα2, and Cα3, a shorthinge section between Cα1 and Cα2, and a short tail piece at theC-terminal end of Cα3. The definition and structure of antibodies arewell known to workers skilled in this art, such as described in, e.g.,Alberts, B. et al., Molecular Biology of the Cell 3^(rd) Edition,Chapter 23, Garland Publishing Inc., New York, N.Y., 1994, and Nezlin,R., The Immunoglobulins. Structure and Function (1998) Academic Press(ISBN 0-12-517970-7).

The C-terminal section of two IgA antibodies, i.e., the tail pieces atthe C-terminal ends of the Cα3 region, can be joined together via aJ-chain to form a dimer. Dimeric IgA has four antigen binding regions;two from each IgA monomer. Typically the four antigen binding regions(and their complementarity determining regions or “CDRs”) are identicalfor reasons such as ease of manufacture. But the antigen binding regionscan, in certain circumstances, be different, e.g., different CDRsbinding different epitopes on the same antigen or event differentantigens (such as in the case of bispecific antibodies). Typically theCDRs of the four antigen binding regions are identical. A secretorychain, sometimes called a secretory component or SC-chain, can beattached to the dimeric IgA antibody. The SC-chain provides increasedresistance to proteolysis especially in the intestinal tract. TheSC-bound dimeric IgA is referred to herein as “secretory IgA” or “SIgA.”

Heavy chain constant regions that qualify as an IgA-class antibody arewell known in the art. Generally the amino acid sequence of the heavychain constant regions of an IgA, regardless of how it is produced(e.g., naturally or recombinantly), corresponds to an amino acidsequence encoded for by an α-gene. In addition, IgA antibodies havecharacteristic antigenic determinants unique to IgA-class antibodies anddifferent from the antigenic determinants of other classes ofantibodies, such as IgG-class antibodies (see, e.g., Nezlin, R., TheImmunoglobulins. Structure and Function (1998) Academic Press (ISBN0-12-517970-7); Lefranc et al., The Immunoglobulin FactsBook, AcademicPress 2001, Chapter 2, (ISBN 0-12-441351-X)). Furthermore, IgAantibodies are the only isotype that is known to specifically bind tothe FcαR (see, e.g., Alberts, B. et al., Molecular Biology of the Cell3^(rd) Edition, Chapter 23, Garland Publishing Inc., New York, N.Y.,1994; Lefranc et al., The Immunoglobulin FactsBook, Academic Press 2001,Chapter 2, (ISBN 0-12-441351-X)).

Accordingly, for purposes of the present invention, the terms “IgAantibody,” “monomeric IgA,” “dimeric IgA” and “SIgA” each refers toantibodies that contain the heavy chain constant regions of an IgA classof immunoglobulin, e.g., which corresponds to an amino acid sequencethat can be encoded for by a genes and which react with an antibodyspecific for the IgA-class heavy chain. The amino acid sequence“corresponds” in that it is identical to, or contains only minorvariations (insertions/deletions/substitutions) from, an amino acidsequence produced by any a gene, an individual human's IgA heavy chainsequence, or a human IgA heavy chain consensus sequence. Indeed,variations can and do exist in the amino acid sequence of the IgA heavychain constant region without moving such antibodies outside of the IgAclass. Examples of such variations can be found in various genomicdatabases such as browser. 1000genomes.org/index.html andensembl.org/index.html. For clarity, because the heavy chain sequence isdeterminative of the Ig class, a recombinant antibody containing the IgAheavy chain constant regions and further containing the antigen bindingregions encoded for by DNA sequences obtained from a known IgG antibodyis still an “IgA antibody.” On the other hand, a secretory IgA antibodymodified to replace the Cα2 heavy chain constant domain (encoded for bythe IgA-specific α-gene) with a Cγ2 heavy chain constant domain (encodedfor by the IgG-specific γ-gene) is not an IgA antibody, and is instead ahybrid IgA/IgG antibody. Such a hybrid is not within the scope of theterms “monomeric IgA,” “dimeric IgA” and “SIgA” antibodies, and thus isnot a secretory IgA antibody according to the invention.

Minor variations of the heavy chain constant regions are permitted onlyto the extent that the overall antibody class, framework, andfunctionality of SIgA is maintained; e.g., J-chain binds to monomers andSC-chain binds to the dimeric structure and provides proteolysisresistance. Such variations include conservative substitutions.Exemplary conservative substitutions are shown in Table 1. The aminoacids in the same block in the second column and preferably in the sameline in the third column may, for example, be substituted for eachother.

TABLE 1 ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

Typical minor variations of the constant regions from the normal ornaturally-occurring sequence involve only conservative changes to theamino acid sequence using the recognized substitutions, insertionsand/or deletions. Generally, the variations (substitutions, insertions,and/or deletions) of a constant domain of the heavy or light chaininvolve no more than 10 and usually no more than 5 amino acid additions,deletions, and/or substitutions (either naturally-occurring orgenetically-engineered), in any Cα1, Cα2, or Cα3 domain or hinge or tailsections in comparison to a normal IgA constant domain. The sum of theseminor variations in the constant domains of the SIgA antibody of theinvention is usually less than 20 amino acids(acid/deletions/substitutions) and often less than 10 or less than 5.

Accordingly, at a minimum, SIgA includes any recognized amino acidsequence that is generally accepted as being within the IgA class. Forexample, information on the structure and function of IgA can be foundin Snoeck et al., Vet. Res. 37; 455-467 (2006) and “Mucosal immunedefense: Immunoglobulin A”, C. S. Kaetzel ed., Springer, New York (2007)ISBN 978-0-387-72231-3. Electronic databases, such as RCSB Protein DataBank, can also establish a known IgA sequence or portion/domain thereof.The constant domains contained in the SIgA antibodies of the inventioncan be human, non-human, or a combination of these. Preferred aremammalian constant regions. Most preferred are human constant regions.

In humans there are two recognized IgA subclasses: IgA1 and IgA2 whichdiffer in the hinge section between the Cα1 and Cα2 domains of the heavychain. In IgA1 this hinge section is relatively long and in naturetypically O-glycosylated. In IgA2 the hinge section is relatively shortand in nature lacks glycosylation. Both IgA1 and IgA2 SIgA antibodiesare usually present in mucosal secretions. In humans, the IgA2 subclasshas three known allotypes: IgA2m(1), IgA2m(2) and IgA2m(n). Unlike thesubclasses, only one specific allotype will be found in a normal healthyindividual. The m(1) allotype is strongly prevalent in the Caucasianpopulation (98%) and varies between 23% and 96% for other populations.The m(2) allotype has a high prevalence in populations of African andAsian descent (50-70%). The m(n) allotype—which is considered to be ahybrid of the m(1) and m(2) allotypes—has been reported to begenetically possible, but has not been actually observed in anypopulation. See Chintalacharuvu et al., Journal of Immunology 152,5299-5304 (1994). Accordingly, SIgA antibodies of the present inventionpreferably contain human IgA heavy chain constant regions of the IgA1 orIgA2 sub-types, including IgA2m(1), IgA2m(2) and IgA2m(n) allotypes, andcombinations thereof (e.g., one constant domain or hinge section from anIgA1 and another constant domain or hinge section from an IgA2).

Typically, the SIgA antibodies of the invention comprise the Cα1 domain,the hinge section, and the -Cα2-Cα3 domains and tail section of an IgAantibody (with or without minor variations), including a human IgA1and/or IgA2 antibody. In this embodiment, the Cal domain, the hingesection, and the -Cα2-Cα3 domains can be of an IgA1, an IgA2m(1)allotype, an IgA2m(2) allotype, or a combination thereof. Amino acidsequences of a human IgA1 heavy chain constant region (SEQ ID NO:10), ahuman IgA2m(1) heavy chain constant region (SEQ ID NO:11), a humanIgA2m(2) heavy chain constant region (SEQ ID NO:12), and a human IgA2(n)heavy chain constant region (corresponding to the Cα1 and Cα2 regions ofan IgA2m(2) and the Cα3 region of an IgA2m(1)) (SEQ ID NO:13) arerespectively shown in FIG. 1.

A modified, shortened, or removed linker/hinge section between Cα1 andCα2 in IgA1 has been reported to increase resistance against proteases(for example see B. W. Senior et al., J. Immunol. 2005; 174: 7792-7799).Such can be incorporated into the SIgA antibodies of the presentinvention.

The J-chain is a protein that attaches to the tail piece of a monomericIgA to join two monomeric IgAs to form a dimer. The J-chain is normallyof mammalian origin, such as human, murine, rat, rabbit, sheep, cow, orgoat origin, but is preferably of human origin. An example of the aminoacid sequence of a human J-chain is set forth in FIG. 4 (SEQ ID NO:20).Usually, the sequence of the mammalian-derived J-chain is the same asthe naturally-occurring sequence, but it can be subject to minorvariations as described above for constant regions generally, e.g., upto 10 amino acid insertions, substitutions, or deletions. The minorvariations do not significantly alter the function of the J-chain, andin particular the ability to join two monomeric IgA antibodies to form adimer and to enable attachment of the SC-chain.

The secretory component, also referred to as “SC” or “SC-chain,” is aprotein that binds to the dimeric IgA framework and imparts increasedresistance against proteolysis upon the antibody to which it is bound.Generally, the secretory component is of mammalian origin, such ashuman, murine, rat, rabbit, sheep, cow, or goat origin, but ispreferably of human origin. The SC is the result of cleavage of thePolymeric IgA-receptor (PIGR) which usually occurs at a specificposition. Some variation can occur in the position of the cleavageresulting in variant forms of SC. Usually, the sequence of themammalian-derived SC-chain is the same as the naturally-occurringsequence, but it can be subject to minor variations as described abovefor constant regions generally, e.g., up to 10 amino acid insertions,substitutions, or deletions. The minor variations do not significantlyalter the function of the secretory component, e.g., the ability tostabilize the SIgA against proteolysis. An example of the amino acidsequence of a human secretory component is set forth in FIG. 5 (SEQ IDNO:21).

Secretory IgA antibodies of the present invention bind to and neutralizea human proinflammatory cytokine or bind to and block a human receptorof a human proinflammatory cytokine. In a preferred embodiment, theantigen binding region of a SIgA according to the invention comprises aheavy and light chain variable region pair, each containinghypervariable regions (CDRs, which directly interact with theproinflammatory cytokine) and the supporting framework regions. The CDRsin each heavy and light chain variable region are separated from eachother and from the constant domain by the framework regions, which serveto maintain the CDRs in the correct binding conformation. In generaleach variable part of an immunoglobulin heavy or light chain contains 3different CDRs and four framework regions. For a more detaileddescription of antibody antigen binding regions, see for example C. A.Janeway et al., “Immunobiology” 6^(th) Edition, Chapter 3, pp 110-115;Garland Science Publishing, New York, 2005 (ISBN 0815341016). Regardingframework regions in particular, see for example WO92/22653 (discussingthat although framework regions do not directly interact with antigen,framework regions can influence binding of the CDRs with antigen, suchas binding strength and/or downstream events).

In these embodiments, the antigen binding regions of the SIgAs of theinvention bind to and neutralize the proinflammatory cytokine or bind toand block a human receptor of a human proinflammatory cytokine.Preferably, the proinflammatory cytokine or receptor is selected fromTNF-alpha (preferably soluble TNF-alpha), IFN-gamma, IFN-alpha, GM-CSF,CXCL10/IP-10, IL-1-beta, IL-1-alpha, IL-4, IL-5, IL-6, IL-12, IL-13,IL-17A, IL-18, IL-20, IL-22, IL-23, IL-1 receptor, IL-2 receptor, IL-4receptor, IL-5 receptor, IL-6 receptor, and IL-17 receptor. Inembodiments, the proinflammatory cytokine is selected from IFN-gamma,IFN-alpha, GM-CSF, CXCL10/IP-10, IL-1-beta, IL-1-alpha, IL-4, IL-5,IL-6, IL-13, IL-17A, IL-18, IL-20, and IL-22.

Secretory IgA antibodies of the present invention specifically andpreferentially bind with high affinity to a corresponding humanproinflammatory cytokine or human receptor of a human proinflammatorycytokine. A variety of protocols for performing binding, competitivebinding, or immuno-radiometric assays to determine the specific bindingcapability of an antibody are well known in the art (see for exampleMaddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassaystypically involve the formation of complexes between the specificprotein and its antibody and the measurement of complex formation (e.g.,binding to or unbinding from the specific protein). Generally, theaffinity of secretory IgA antibodies of the invention is at leasttwo-fold, at least 10-fold, at least 50-fold, at least 100-fold, or atleast 1000-fold or greater than the affinity of the antibodies for anon-specific protein such as, for instance, BSA or casein. Typically thesecretory IgA antibodies of the present invention exhibit a bindingaffinity constant (K_(D)) with respect to the proinflammatory cytokineor proinflammatory cytokine receptor of 10⁻⁷ M or lower, preferably 10⁻⁸M, 10⁻⁹ M, 10⁺¹⁰ M, 10⁻¹¹ M or 10⁻¹²M or lower.

In embodiments, secretory IgA antibodies of the present inventionneutralize a proinflammatory cytokine to which it is bound. For thepresent invention, the term “neutralizes” means inhibits/reduces theeffect of the cytokine to some degree, such as by at least 30%, at least35%, at least 40%, and at least 45%. Typically the inhibition/reductionin the effect of the cytokine at least 50%. In these embodiments, thesecretory IgA antibodies of the present invention preferablyinhibit/reduce the proinflammatory effect of a cytokine to which it isbound by at least 50%, such as by at least 55%, at least 60%, at least65%, and at least 70%, and more preferably by at least 75%, at least80%, at least 85%, at least 90%, at least 95%, and at least 98%.

In embodiments, SIgA antibodies of the present invention blocks aproinflammatory cytokine receptor to which it is bound. For the presentinvention, the term “blocks” means inhibits/reduces the effect of thereceptor to some degree, such as by at least 30%, at least 35%, at least40%, and at least 45%. Typically the inhibition/reduction in the effectof the receptor at least 50%. In these embodiments, the secretory IgAantibodies of the present invention preferably inhibit/reduce theproinflammatory effect of a receptor to which it is bound by at least50%, such as by at least 55%, at least 60%, at least 65%, and at least70%, and more preferably by at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, and at least 98%.

The amino acid sequence of the antigen binding region, and in particularthe CDRs thereof, is determined by the epitope to which it binds. Theamino acid sequence of the antigen binding region can be novel or can beobtained from existing anti-proinflammatory-cytokine antibodies. Methodsfor obtaining novel antigen binding sequences are well known in the art.See for example Mary A. Ritter and Heather M. Ladyman (Eds.) “Monoclonalantibodies: production, engineering, and clinical application”,Cambridge University Press (1995), ISBN 0521425034, Zhiqiang An“Therapeutic Monoclonal Antibodies: From Bench to Clinic” Wiley, NewYork (2009), ISBN: 0470117915, and Christopher Dean and Philip Shepherd(Ed.) “Monoclonal Antibodies: A Practical Approach” Oxford UniversityPress, USA (2000), ISBN 0199637229.

There are many therapeutic antibodies in clinical trials or on themarket that function to neutralize proinflammatory cytokines, or toblock proinflammatory cytokines receptors, to treat various inflammatorydiseases, and the antigen binding regions (and CDRS thereof) of theseknown antibodies are suitable for use in the sectretory IgA antibodiesof the present invention. Examples of such known antibodies, along withthe neutralized cytokines and inflammatory diseases treated, aresummarized in the following Table 2. See, for example, Nature Reviews,vol. 10, pp. 301-316 (2010); Nature Medicine, vol. 18, pp. 736-749(2012); Nature Biotechnology, vol. 30, pp. 475-477 (2012);Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, vol. 8,pp. 51-71 (2009); F1000.com/Reports/Biology/content/1/70, F1000 BiologyReports, 1:70 (2009);landesbioscience.com/journals/mabs/ReichertMABS4-1.pdf, mAbs 4:1, pp.1-3 (2012); landesbioscience.com/journals/mabs/article/13895/, mAbs 3:1,pp. 76-99 (2011); clinicaltrials.gov (generally), andclinicaltrialsregister.eu/ (generally).

TABLE 2 Proinflammatory Cytokine Target Antibody Inflammatory DiseaseTNF-alpha adalimumab (Humira; Abbott); infliximab Rheumatoid arthritis(RA); (Remicade; Janssen); golimumab (Simponi; inflammatory boweldisease (IBD, Centocor); certolizumab pegol (Cimzia; UCB); whichincludes Crohn's disease ozoralizumab (Pfizer); ATN-192 (PF-05230905;and ulcerative colitis); psoriasis; Pfizer); ART621 (Arana). psoriaticarthritis; ankylosing spondylitis; juvenile idiopathic arthritis (JIA);uveitis; (eosinophilic allergic) asthma; and/or Alzheimer's disease.IFN-gamma AMG 811 (Amgen). Psoriasis. IFN-alpha sifalimumab (MedImmune).Psoriasis. GM-CSF MOR103 (MorphoSys AG). RA; multiple sclerosis (MS).CXCL10/IP-10 MDX-1100 (Medarex). RA; IBD. IL-1-beta Canakinumab (Ilaris;Novartis); rilonacept RA; JIA; type II diabetes; uveitis; (Arcalyst;Regeneron); gevokizumab; (XOMA). systemic sclerosis (scleroderma).IL-1-alpha TrueHuman RA-18C3 (XBiotech). Psoriasis. IL-4 pascolizumab(PDL). (Eosinophilic allergic) asthma. IL-5 reslizumab (Cephalon);mepolizumab (GSK); (Eosinophilic allergic) asthma. SCH55700 (Celltech).IL-6 sirukumab (Centocor); olokizumab (UCB); PF- RA; IBD; lupusnephritis. 04236921 (Pfizer); C326 (Avidia). IL-12/23 (p40) ustekinumab(Stelara; Janssen); briakinumab IBD; psoriasis; psoriatic arthritis;(Abbott). ankylosing spondylitis; MS. IL-13 QAX576 (Novartis);tralokinumab IBD; (eosinophilic allergic) (MedImmune); anrukinzumab(Pfizer); asthma; allergic rhinitis. lebrikizumab (Genentech); IMA-026(Pfizer). IL-17A secukinumab (Novartis); ixekizumab (Eli Lilly). RA;IBS; psoriasis; psoriatic arthritis; ankylosing spondylitis; uveitis;MS; (eosinophilic allergic) asthma. IL-18 GSK1070806 (GSK). IBD; type IIdiabetes. IL-20 NN8226 (Novo Nordisk). RA; psoriasis. IL-22 fezakinumab(Pfizer). RA; psoriasis. IL-23 (p19) MK-3222 (Merck); CNTO-1959(Centocor); Psoriasis; IBD. AMG-139 (Amgen). IL-1 receptor GSK1827771(GSK); AMG-108 (Amgen). RA. IL-2 receptor daclizumab (Zenapax; Roche);basiliximab IBD; psoriasis; JIA; uveitis; MS; (Simulect; Novartis).(eosinophilic allergic) asthma. IL-4 receptor AMG-317 (Amgen).(Eosinophilic allergic) asthma. IL-5 receptor benralizumab (MedImmune).(Eosinophilic allergic) asthma. IL-6 receptor tocilizumab (Actemra;Roche); ALX-0061 RA; ankylosing spondylitis; JIA. (Ablynx); sarilumab(Regeneron). IL-17 receptor brodalumab (Amgen). RA; IBD; psoriasis;(eosinophilic allergic) asthma; psoriatic arthritis.

In embodiments, a secretory IgA antibody according to the inventioncomprises CDR sequences that are identical to the CDR sequences of anantibody selected from adalimumab, infliximab, golimumab, certolizumabpegol, ozoralizumab, sifalimumab, canakinumab, gevokizumab,pascolizumab, reslizumab, mepolizumab, sirukumab, olokizumab,ustekinumab, briakinumab, tralokinumab, anrukinzumab, lebrikizumab,secukinumab, ixekizumab, and fezakinumab.

The CDR sequences for these antibodies are publicly available. Forexample, for adalimumab see WO8907452, for infliximab see CAS RegistryNo. 170277-31-3 (entered STN 17 Nov. 1995), for golimumab see CASRegistry No. 476181-74-5 (entered STN 13 Dec. 2002) and WO2007028106,for certolizumab pegol see CAS Registry No. 428863-50-7 (entered STN 12Jun. 2002), for ozoralizumab see CAS Registry No. 1167985-17-2 (enteredSTN 24 Jul. 2009), for sifalimumab see CAS Registry No. 1006877-43-3(entered STN 6 Mar. 2008) and WO2012162367, for canakinumab see CASRegistry No. 914613-48-2 (entered STN 4 Dec. 2006) and EP1313769, forgevokizumab see CAS Registry No. 1129435-60-4 (entered STN 30 Mar. 2009)and WO2009086003, for pascolizumab see CAS Registry No. 331243-22-2(entered STN 13 Apr. 2001), for reslizumab see CAS Registry No.241473-69-8 (entered STN 25 Sep. 1999) and U.S. Pat. No. 6,451,982, formepolizumab see CAS Registry No. 196078-29-2 (entered STN 24 Oct. 1997),for sirukumab see CAS Registry No. 1194585-53-9 (entered STN 1 Dec.2009), for olokizumab see CAS Registry No. 1007223-17-7 (entered STN 10Mar. 2008), for ustekinumab see CAS Registry No. 815610-63-0 (enteredSTN 18 Jan. 2005) and EP1309692, for briakinumab see CAS Registry No.339308-60-0 (entered STN 4 Jun. 2001), for tralokinumab see CAS RegistryNo. 1044515-88-9 (entered STN 29 Aug. 2008) and WO2005007699, foranrukinzumab see CAS Registry No. 910649-32-0 (entered STN 18 Oct.2006), for lebrikizumab see CAS Registry No. 953400-68-5 (entered STN14Nov. 2007), for secukinumab see EP1776142, for ixekizumab see CASRegistry No. 1143503-69-8 (entered STN 7 May 2009), and for fezakinumabsee CAS Registry No. 1007106-86-6 (entered STN 7 Mar. 2008). Of course,these are not necessarily the only source for the CDR sequences, e.g.,some CDR sequences can be obtained from other patent and non-patentliterature, and commercially-available antibodies can be purchased andsequenced.

For example, a secretory IgA antibody according to the invention cancomprise CDR sequences that are identical to the CDR sequences of anantibody selected from golimumab, certolizumab pegol, ozoralizumab,sifalimumab, canakinumab, gevokizumab, pascolizumab, reslizumab,mepolizumab, sirukumab, olokizumab, briakinumab, tralokinumab,anrukinzumab, lebrikizumab, secukinumab, ixekizumab, and fezakinumab. Asanother example, a secretory IgA antibody according to the invention cancomprise CDR sequences that are identical to the CDR sequences of anantibody selected from ozoralizumab, sifalimumab, canakinumab,gevokizumab, pascolizumab, reslizumab, mepolizumab, sirukumab,olokizumab, tralokinumab, anrukinzumab, lebrikizumab, secukinumab,ixekizumab, and fezakinumab.

In embodiments a secretory IgA antibody according to the inventioncomprises CDR sequences that are identical to the CDR sequences of anantibody selected from daclizumab, basiliximab, benralizumab,tocilizumab, sarilumab, brodalumab, and omalizumab. The CDR sequencesfor these antibodies are publicly available. For example, for daclizumabsee CAS Registry No. 152923-56-3 (entered STN 10 Feb. 1994) andEP932415, for basiliximab see CAS Registry No. 179045-86-4 (entered STN1 Aug. 1996) and EP0449769, for benralizumab see CAS Registry No.1044511-01-4 (entered STN 29 Aug. 2008), for tocilizumab see CASRegistry No. 375823-41-9 (entered STN 17 Dec. 2001) and EP0783893, forsarilumab see CAS Registry No. 1189541-98-7 (entered STN 22 Oct. 2009),for brodalumab see CAS Registry No. 1174395-19-7 (entered STN 17 Aug.2009), and for omalizumab see CAS Registry No. 242138-07-4 (entered STN28 Sep. 1999) and EP0602126. Of course, these are not necessarily theonly source for the CDR sequences, e.g., some CDR sequences can beobtained from other patent and non-patent literature, andcommercially-available antibodies can be purchased and sequenced.

In still other embodiments, antigen binding regions of secretory IgAantibodies of the invention are obtained from other and/or novelanti-proinflammatory cytokine (or anti-proinflammatory cytokinereceptor) antibodies. Methods for obtaining antibodies against specificantigens are well known in the art and can be used to obtain suitableinflammatory cytokine-binding variable regions. See for example Mary A.Ritter and Heather M. Ladyman (Eds.) “Monoclonal antibodies: production,engineering, and clinical application”, Cambridge University Press(1995), ISBN 0521425034, Zhiqiang An “Therapeutic Monoclonal Antibodies:From Bench to Clinic” Wiley, New York (2009), ISBN: 0470117915, andChristopher Dean and Philip Shepherd (Ed.) “Monoclonal Antibodies: APractical Approach” Oxford University Press, USA (2000), ISBN0199637229.

Secretory IgA antibodies of the invention can be non-human antibodies,chimeric antibodies, humanized antibodies, human antibodies, or othermixes of human and non-human sequences/regions. See, e.g., Yamashita etal. Cytotechnology 55: 55-60 (2007). A chimeric antibody is an antibodyhaving an antigen binding region (CDRs and framework) originating from afirst species (typically a mouse) and heavy chain constant regionsoriginating from a second species (typically a human). A humanizedantibody is a human antibody onto which non-human (typically murine)CDRs have been grafted. In the humanized antibody, certain humansupporting framework amino acid residues can be replaced with theircounterparts from the non-human parent antibody. Such an antibodycontaining certain non-human framework residues is still a humanizedantibody. See, e.g., WO92/22653. In the humanized antibodies, thesequence of the supporting framework into which the non-human CDRs aregrafted can be obtained from any human isotype/class, preferentiallyfrom IgG or IgA, and may be modified to improve the properties thereof(e.g., antigen binding and/or downstream effects). A human antibody isfully-human, containing only human constant and variable regions, i.e.,having only human heavy and light chains (derivable from human genomicsequences by naturally-occurring recombination and mutation processes,consensus sequences, etc.). Likewise, a non-human antibody contains onlynon-human constant and variable regions, i.e., having only non-humanheavy and light chains.

The secretory IgA antibodies of the invention may contain additionalatoms, moieties, molecules, and/or modifications beyond the dimeric IgA,J-chain, and SC-chain. For example, the secretory IgA antibodies of theinvention may be PEGylated or glycosylated (or aglycosylated) in variousorientations and/or amounts. The location, attachment, amount, andstructure of attached glycans found in naturally occurring antibodiesshows substantial variability and mainly depends on the source of theglycoprotein (i.e., the type of cell producing the glycoprotein), but isalso influenced by growing conditions (i.e., feed and environmentalconditions). The secretory IgA antibodies of the present invention arenot limited to any specific form of glycosylation and specificallyinclude non-glycosylated proteins, partially or fully deglycosylatedproteins, variants obtained by genetic or other manipulation of theglycosylation system of the producing cell, and variants with chemicallyor enzymatically modified glycans. The secretory IgA antibodies of theinvention can be glycoproteins with glycosylation patterns native toplant, mammalian (human), or insect cells. Additionally the antibodiesof the invention may be conjugated with (fluorescent) markers ortherapeutic agents, etc. (see, e.g., Lash, A. “Making the case forantibody-drug conjugates;” In Vivo: The Business & Medicine Report; vol.28, No. 11, pp. 32-39 (December 2010) (www.ElsevierBI.com).

It is recognized that antibodies having more than one glycosylation sitecan have the same glycan species attached to each glycosylation site, orcan have different glycan species attached to different glycosylationsites. In this manner, different patterns of glycan attachment yielddifferent glycoforms of a glycoprotein. Monomeric IgA1 antibodies havetwo conserved N-glycosylation sites (per chain): one on the CH2 regionand one on the tailpiece. Monomeric IgA2 antibodies have an additionaltwo or three N-glycosylation sites (per chain). Furthermore, the J-chainof dimeric IgA has one conserved N-glycosylation site, and the secretorycomponent of secretory IgA has 7 conserved N-glycosylation sites.

The terms “N-glycan(s)” and “N-linked glycan(s)” are usedinterchangeably and refer to an N-linked oligosaccharide, e.g., one thatis or was attached by an N-acetylglucosamine (GlcNAc) residue linked tothe amide nitrogen of an asparagine residue in a protein. Thepredominant sugars found on glycoproteins are glucose (Glu), galactose(Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc),N-acetylglucosamine (GlcNAc), and sialic acid (e.g., N-acetyl-neuraminicacid (NeuAc)). The processing of the sugar groups occursco-translationally in the lumen of the ER and continues in the Golgiapparatus for N-linked glycoproteins.

For the purposes of the present invention, the term “G2 glycan,” “G2glycan species,” and “G2 glycan structure” are used interchangeably andrefer to an N-linked glycan having the GlcNAc2Man3GlcNAc2Gal2 structure,in which two terminal galactose (Gal) sugar residues are present. Forthe purposes of the present invention, the term “G1 glycan,” “G1 glycanspecies,” and “G1 glycan structure” are used interchangeably and referto an N-linked glycan having the GlcNAc2Man3GlcNAc2Gal structure, inwhich only one terminal galactose (Gal) sugar residue is present. Forthe purposes of the present invention, the term “G0 glycan,” “G0 glycanspecies,” and “G0 glycan structure” are used interchangeably and referto an N-linked glycan having the GlcNAc2Man3GlcNAc2 structure, in whichno terminal galactose (Gal) sugar residues are present.

For the purposes of the present invention, the term “high-mannoseglycan,” high-mannose glycan species,” and “high-mannose glycanstructure” are used interchangeably and refer to an N-linked glycanhaving the GlcNAc2ManX structure, wherein X is a whole number greaterthan three, such as 5, 6, 7, 8, or 9. For the purposes of the presentinvention, the term “Man5 glycan,” Man5 glycan species,” and “Man5glycan structure” are used interchangeably and refer to an N-linkedglycan having the GlcNAc2Man5 structure. The same is applicable for theterms Man6 glycan (species; glycan structure), Man7 glycan (species;glycan structure), Man8 glycan (species; glycan structure), Man 9 glycan(species; glycan structure), etc.

In mammals, naturally-occurring N-glycans contain a fucose (Fuc) residueattached to the GlcNAc2Man3 core structure by an α1,6 linkage. Inplants, naturally-occurring N-glycans contain a fucose (Fuc) residueattached to the GlcNAc2Man3 core structure by an α1,3 linkage andfurther contain a xylose (Xyl) residue attached to the GlcNAc2Man3 corestructure by a β1,2 linkage. For the purposes of the present invention,a G0 glycan containing the mammalian α1,6-linked Fuc residue attached tothe GlcNAc2Man3 core structure is referred to as a “G0F<6> glycan.” Forthe purposes of the present invention, a G0 glycan containing the plantα1,3-linked Fuc residue attached to the GlcNAc2Man3 core structure isreferred to as a “G0F<3> glycan,” a G0 glycan containing the plantβ1,2-linked Xyl residue attached to the GlcNAc2Man3 core structure isreferred to herein as a “G0X glycan,” and a G0 glycan containing each ofthe plant α1,3-linked Fuc residue and the plant β1,2-linked Xyl residueattached to the GlcNAc2Man3 core structure is referred to herein as a“G0XF<3> glycan.” In an embodiment, the invention relates to a secretoryIgA antibody, or a population of secretory IgA antibodies, in whichsubstantially all N-glycans lack Fuc and Xyl residues.

The present invention also relates to a composition comprising aplurality of secretory IgA antibodies containing multiple N-glycans,such as two or more different N-glycans. In embodiments, the pluralityof secretory IgA antibodies contains at least about 30% G0 glycans(preferably G0 glycans lacking Fuc and Xyl residues) relative to thetotal amount of N-glycans in the population. In embodiments, theplurality of secretory IgA antibodies contains at least about 25%high-mannose glycans (e.g., Man5, Man6, Man7, Man8, and/or Man9 glycans)relative to the total amount of N-glycans in the population. Inembodiments, G0 glycans (preferably G0 glycans lacking Fuc and Xylresidues) and high-mannose glycans (e.g., Man5, Man6, Man7, Man8, and/orMan9 glycans) together are the majority of glycans present in theplurality of secretory IgA antibodies, such as at least 70% relative tothe total amount of N-glycans in the plurality of secretory IgAantibodies.

The glycoform of an antibody, and the nature of glycan species, can bedetermined by measuring the glycosylation profile thereof. The term“glycosylation profile” means the characteristic fingerprint of therepresentative N-glycan species that have been released from anantibody, either enzymatically or chemically, and then analyzed fortheir carbohydrate structure, for example, using LC-HPLC, or MALDI-TOFMS, and the like. See, for example, the review in Current AnalyticalChemistry, Vol. 1, No. 1 (2005), pp. 28-57.” For more information onglycosylation of therapeutic antibodies, see, e.g., Fernandes et al.,Eur. Biopharm. Rev., Summer 2005, pp. 106-110; Jefferis, NatureReviews/Drug Discovery, vol. 8, Mar. 2009, pp. 226-234.

The SIgA antibodies of the present invention are preferably monoclonalantibodies. A “monoclonal antibody” refers to a population or collectionof antibodies that are substantially identical because they were allproduced by clones of a single cell. For the present invention, amonoclonal SIgA is a SIgA containing monoclonal monomeric IgAantibodies. Preferably, a monoclonal SIgA contains monomeric IgAantibodies, a J-Chain, and an SC-chain that were all produced by a cloneof a single cell.

The antibodies of the present invention are often isolated or in anisolated form. As used herein, the terms “isolate,” “isolating” and“isolation” refer to separating the antibody from its productionenvironment. The extent of separation is generally at least 50%, but isfrequently at least 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% (w/w).When the SIgA antibody of the present invention is produced in a cell,which is the typical process, the separation refers to separating theantibody from the host cells and native host cell proteins. Isolation isthus related to purification. Preferably the antibody of the presentinvention in isolated form has removed, or been separated from, at least90%, more typically at least 99% (w/w) of the host cell proteins of theoriginal composition.

Various compositions that contain the secretory IgA antibodies of thepresent invention, whether in an isolated form or not, are alsocontemplated as being part of the present invention. For instance,compositions that contain low amounts of incomplete secretory IgAantibodies are often desirable. With respect to the amount of secretoryIgA antibodies in the composition, the amount of dimer IgA (no SC-chain)is desirably less than 50%, more desirably less than 25% and often lessthan 10%. Thus in a composition that contained 10 mg of secretory IgA,the amount of non-SC-chain dimeric IgA would preferably be less than 1mg, i.e., less than 10%. The same is true for monomeric IgA: the contentof IgA monomers is desirably less than 50% the amount of secretory IgA,more desirably less than 25% and often less than 10%. In someembodiments, the combined amount of dimer IgA (i.e., no SC-chain) andmonomer IgA is less than 25% of the amount of secretory IgA in thecomposition, often less than 10%, and even less than 5%. The aboveamounts apply to both isolated and non-isolated forms of secretory IgAcompositions. Accordingly, the low relative amounts of incompletesecretory IgA can be a result of the expression system (nativelow-production of incomplete secretory IgA), the result of someseparation or purification that removes incomplete secretory IgAantibodies, or both.

Purified secretory IgA compositions are also useful. A purifiedsecretory IgA (composition) contains a secretory IgA antibody of thepresent invention in an amount of at least 85%, often at least 90%, moreoften at least 95%, and preferably at least 97%, 98%, or 99%, based onthe total soluble protein content. The purified composition can be asolid, such as a lyophilized product, or a liquid. A typical liquid formcontains no solids, e.g., no insoluble cell wall materials, and is oftenbased on water as the main or sole solvent and optionally containingsalts, pH adjusting agents, or buffers. A purified liquid compositiongenerally contains the secretory IgA antibody of the invention in aconcentration of 50 μg/ml or more, often at least 100 μg/ml, preferablyat least 1 mg/ml.

Production of Proteins of the Present Invention

The SIgA antibodies of the invention can be produced using recombinanttechniques. Although several expression systems are known, includingCHO, HEK, yeast, tobacco, etc., the use of duckweed as the host cell hasbeen found to be advantageous for the production of SIgAs. Other planthost cells, namely tobacco and lettuce, tend to give very low expressionrates of the desired SIgA and typically render impractical a measurablerecovery of the antibody. Similarly, CHO cells also tend to give lowresults. HEK generally have higher titers than CHO cells, but havecertain production and regulatory disadvantages. Accordingly, duckweedis, surprisingly, a convenient host cell for expressing secretory IgAsof the present invention.

Generally, a genetically modified duckweed is a known expression systemfor producing various proteins (see U.S. Pat. No. 6,040,498), includingfor the production of monoclonal antibodies (see U.S. Pat. No.7,632,983). Duckweed is the common name for the members of themonocotyledonous family Lemnaceae. The five known genera and 38 speciesof Lemnaceae are all small, free-floating, fresh-water plants whosegeographical range spans the entire globe: genus Lemna (L.aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L.minor, L. minuscula, L. obscura, L. perpusilla, L. tenera, L. trisulca,L. turionifera, L. valdiviana); genus Spirodela (S. intermedia, S.polyrrhiza); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina,Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa.globosa, Wa. microscopica, Wa. neglecta) genus Wolfiella (Wl. caudata,Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda,Wl. rotunda, and Wl. neotropica), and genus Landoltia (L. punctata). Forclarity, the term “duckweed” as used in the present invention includesthe foregoing species, genetically modified variants thereof (e.g.,modified to control glycosylation, secretion, etc.), and any othergenera or species of Lemnaceae, if they exist, optionally in agenetically modified form. Typically the genus Lemna is preferred,especially the species L. minor and L. gibba in natural or geneticallymodified forms. Also, the use of the term “duckweed,” or any genus orspecies thereof, is meant to include individual plant cell(s), nodules,as well as whole plants including mature plants having root and fronds,unless otherwise indicated by context or express statement.

Recombinant production of sIgAs in duckweed requires transformation ofduckweed, either transiently or stably. For production purposes, astable transformation, wherein the nucleic acid sequences and/or genesneeded to produce the desired SIgA have been operably introduced intothe genome of a duckweed, is typically preferred. Stable transgenesis induckweed can be obtained by different techniques as described in U.S.Pat. Nos. 6,040,498 and 7,161,064 to Stomp et al. Briefly stableduckweed transgenesis can be achieved by DNA-coated particlebombardment, electroporation, and Agrobacterium spp.-mediatedtransformation. Preferably, transgenesis of duckweed is performed byusing A. tumefaciens-mediated transformation. Briefly,Agrobacterium-mediated transformation is carried out bydedifferentiating fully grown duckweed plants or tissues, preferablytissues of meristematic origin, into calli. Callus induction is carriedout by growing duckweed in medium containing plant growth regulators andsupplements. Calli can/will re-differentiate into organized nodules.Both nodules or calli can be infected with Agrobacterium, according tothe procedure described in U.S. Pat. Nos. 6,040,498 and 7,161,064 toStomp et al. Regeneration of plants from infected calli/nodules andconcomitant selection for transformants by applying the desiredselective pressure results in the isolation of transgenic duckweed linescarrying the exogenous DNA of interest.

Construct for expression of SIgAs, to be used for transformation ofduckweed, can be produced by using standard techniques for example, thetechniques described in Sambrook & Russell, Molecular Cloning: ALaboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, NY (2001)and Ausubel et al, Current Protocols in Molecular Biology (GreenePublishing Associates and Wiley Interscience, NY (1989)). Vectors fortransformation of duckweed have been described elsewhere, such as inU.S. Pat. Nos. 6,040,498 and 7,161,064 to Stomp et al. Preferably, an A.tumefaciens binary vector (generated, for example, by standard cloningin E. coli) is used to first transform A. tumefaciens; the transgenicline obtained can then be employed to transform duckweed. Preferably,such vectors contain multiple resistance genes, to allow for selectionin bacteria and in duckweed. Genes for bacterial selection are known inthe art. Suitable resistance genes for selection in plants have beendescribed in U.S. Pat. Nos. 6,040,498 and 7,161,064 to Stomp et al., andinclude gentamycin and kanamycin.

For expression of SIgAs, multiple transformations can be performed withseparate vectors including different cassette coding for the J-chain,the SC-chain, the antibody H chain and L chain. In a preferredembodiment, a single vector is used for transformation that contains 4cassettes each encoding for one of the structural subunit of the SiGA(namely, H chain, L chain, SC-chain and J-chain). Construction ofvectors containing multiple expression cassette for antibody expressionhave been described in U.S. Pat. No. 7,632,983 to Dickey et al.

In one embodiment, expression of the cassettes is driven by individualpromoters. Examples of suitable promoter can be found in US patents U.S.Pat. No. 4,771,002 to Stanton, U.S. Pat. No. 5,428,147 to Barker et al.,U.S. Pat. No. 7,622,573 & U.S. Pat. No. 8,034,916 to Dickey et al.,disclosures of which are incorporated herein by references. Mostpreferably, four different promoters are used for each expressioncassette (such as the chimeric A. tumefaciens octopine and mannopinesynthase promoter, the L. minor polyubiquitin promoter (LmUbq), Lemnaaequinoctialis polyubiquitin promoter (LaUbq) and Spirodela polyrrhizapolyubiquitin promoter (SpUbq). In a preferred embodiment, theexpression vector includes cassettes coding for all 4 of the SIgAcomponents, i.e. J-chain, SC-chain, H-chain and L-chain. In even morepreferred embodiment, each of the 4 cassettes is driven by a differentpromoter. In a different embodiment, the constructs are driven by, heatshock gene promoters, cold-induced promoters, drought-inducible genepromoters, pathogen-inducible gene promoters, wound-inducible genepromoters, and light/dark-inducible gene promoters, promoters from genesinduced by abscissic acid, auxins, cytokinins, and gibberellic acid, asdescribed in U.S. Pat. No. 7,632,983 to Dickey et al.

In an advantageous embodiment, the vectors used for expression include,5′ of the coding sequence of the expression cassette, a signal peptidesequence placed in frame with the N-terminal portion of the protein ofinterest. Such signal peptide sequence interacts with a receptor proteinon the membrane of the endoplasmic reticulum (ER) to direct thetranslocation of the growing polypeptide chain across the membrane andinto the endoplasmic reticulum for secretion from the cell. Presence ofthe signal peptide sequence ensures efficient secretion into theextracellular space. This signal peptide is generally cleaved from theprecursor polypeptide to produce a mature polypeptide lacking the signalpeptide. Suitable signal peptide include the Arabidopsis thalianachitinase signal peptide, the Oryza sativa α-amylase signal peptide, orany other suitable duckweed signal peptide sequences, as described inU.S. Pat. No. 7,632,983 to Dickey et al. In a most preferred embodiment,the sequence of the signal peptide used in the O. sativa α-amylasesignal peptide. In some embodiments of the present invention, thesecreted SIgAs are retained within the apoplast, the region between theplasma membrane and the cell wall. In other embodiments, the polypeptidediffuses across the cell wall of the plant host cell into the externalenvironment/media.

Other suitable nucleotide sequences including enhancers, 5′ leadersequences, such as the leader sequence of L. gibbaribulose-bis-phosphate carboxylase small subunit 5B gene, 3′ UTRsequences, introns, enhancers, “ACC” and “ACA” trinucleotides to beintroduced directly upstream of the translation initiation codon of thenucleotide sequence of interest can be used to improve expression asdisclosed in the art and in U.S. Pat. Nos. 6,040,498 and 7,161,064 toStomp et al as well as U.S. Pat. No. 7,622,573 & U.S. Pat. Nos.8,034,916 7,632,983 to Dickey et al, disclosures of which are allincorporated by reference herein.

For example, the expression from the transgenic lines obtained can beimproved by optimizing the codon distribution of the encoded proteinsfor expression in duckweed. Duckweed-preferred codons, as used herein,refers to codons that have a frequency of codon usage in duckweed ofgreater than 17%. Likewise the codons can be optimized for expression inL. minor or L. gibba. In each case the codons have a frequency of codonusage of greater than 17%. Duckweed and Lemna ssp. codon optimization isknown in the art and is carried out, e.g. as described in U.S. Pat. No.7,632,983 to Dickey et al.

Another option is to modify the glycosylation profile of the duckweed.The stably transformed duckweed can also contain a genetic modificationthat alters the glycan profiles. For example, the N-glycans of the SIgAcan be expressed with reduced levels of fucose and xylose residue,preferably less than 10%, more preferably less than 1%. Thismodification from natural glycan profile can be achieved by severaltechniques, including knocking out endogenous α1,3-fucosyltransferase(FucT) and β1,2-xylosyltransferase (XylT), or otherwise inhibiting theirtranscription of the gene/expression or enzymatic activity. In apreferred embodiment, the duckweed is transformed with at least onerecombinant nucleotide construct that provides for the inhibition ofexpression of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase(XylT) in a plant. In a more preferred embodiment, these constructstriggers RNA interference targeting the mRNAs of α1,3-fucosyltransferase(FucT) and β1,2-xylosyltransferase (XylT). In an even more preferredembodiment, the construct is a RNA hairpin construct. These methods foraltering the N-glycosylation pattern of proteins in duckweed are knownin the art and are described in U.S. Pat. No. 7,884,264 to Dickey et al.The use of the RNA hairpin construct can be advantageous for obtaining aglycan profile where at least 30% of the N-glycans are G0 glycanslacking Fuc and Xyl residues and/or where the combination of G0 glycanslacking Fuc and Xyl plus high-mannose glycans are at least 70% relativeto the total amount of N-glycans in the plurality of secretory IgAantibodies.

Once the transformed duckweed is obtained, the genetic modification willcause the duckweed to express the desired SIgA antibody during itsotherwise normal metabolic activity. The term “express” and itsgrammatical variants refers to the biosynthesis of the SIgA antibody,which includes the transcription, translation, and assembly of theantibody by the duckweed. Generally this entails providing anenvironment to keep the duckweed alive and/or to promote growth; e.g.,providing light (natural and/or artificial) and a liquid mediumtypically based on water. Providing this environment is often referredto as “culturing” the duckweed. Methods of culturing duckweed includingthe media, supplements (if any), conditions, etc., are known in the artand have been disclosed in, e.g., U.S. Pat. Nos. 6,040,498; 7,161,064;and 7,632983; and references cited therein, respectively.

Culturing of transgenic duckweed of the invention can be performed intransparent vials, flask, culture bags, or any other container capableof supporting growth using defined media. In some embodiments of theinvention large scale growth of duckweed, necessary to achieveindustrial production levels, is carried out in bioreactortailor-designed for growth of duckweed. In a preferred embodiment,duckweed bioreactors, which can be inoculated aseptically, supportaseptic growth of duckweed. In even more preferred embodiments, abioreactor can be directly connected to harvest bag to separate themedia from the plant material, either of which can then be piped intodownstream purification steps. Suitable bioreactors, methods/devices toinoculate them aseptically, and aseptic harvest bags are described inU.S. Pat. No. 7,176,024 to Branson et al. or in US application2010/209966 To Everett et al.

Following expression of the fully formed SIgA antibody, recovery of theantibody from the duckweed and/or the culture media is often desired.The first step, generally, is to separate the SIgA antibody from theduckweed. If the antibody is secreted and diffuses into the culturemedia, then a simple filter can separate the crude antibody product fromthe duckweed. Typically, however, the fully formed SIgA antibody isretained within the duckweed's apoplast. Separation in this casegenerally requires extraction.

Extraction of secreted SIgAs typically involves a homogenization step todisrupt the plant material and allow for release of the secreted SIgAfrom the apoplast into the homogenization buffer; also referred to asextraction buffer or extraction media. Homogenization buffers andtechniques are known in the art. Small scale homogenization can beperformed manually, such as by using mortar-and-pestle crushing, and thelike. Larger scale homogenization is preferably performed using amechanical mixer, typically a high shear mixer such as a Silverson 275UHS mixer, or similar apparatuses. The buffer is typically an aqueousphosphate buffer composition though such is not required. The buffer maycontain additional functional ingredients as is known in the art. Forexample, to reduce proteolysis by metallated proteases, EDTA may beadded to the extraction buffer, typically in amounts from 1 to 20 mM,including 5 to 10 mM. Also, one or more anti-oxidants, such as ascorbicacid, sodium metabisulfite, benzyl alcohol, benzoic acid, and the like,may be added during the homogenization process. Homogenization isgenerally followed by centrifugation and filtration to obtain a buffersolution that contains the SIgA antibodies and other soluble proteins.

To remove some of the unwanted soluble proteins, homogenization is oftenfollowed by clarification; a step that seeks to remove certain naturallyabundant impurities including (host cell proteins), such as RuBiSco, aswell as non-proteinaceous impurities, such as tannins. This is usuallyachieved by acidic precipitation. For example, clarification can beperformed by adjusting the pH of the filtrated homogenate to 4.5,followed by centrifugation (such as for 30 min at 12000), neutralizationto pH 7.4, and an additional filtration step. In a preferred embodiment,pH adjustments are performed using 1 M citric acid pH 1.5, or 1M sodiumacetate for acidification and 2M tris-base for neutralization, thoughother suitable pH adjusting agents can also be used instead of or inaddition to such agents. Filtration is performed as known in the art,often by using a 0.22 μm filter.

The recovery of the SIgA antibodies from duckweed may end with theextraction buffer or the clarified material. However, for some uses,purification of the antibody is desired. Purification can be performedusing known methods and techniques and generally comprises subjectingthe clarified material to affinity chromatography (AC), size exclusionchromatography (SEC), and optional polishing steps. For efficiency, ACusually precedes SEC, though such is not required.

Methods of using affinity chromatography (AC) as a purification step toremove contaminant proteins and impurities are known in the art and aredescribed in Process Scale Purification of Antibodies (2009), Edited byU. Gottschalk, J. Wiley and son, Hoboken, N.J., and references citedtherein. Usually the SIgA antibody is bound to the affinity resinmaterial while one or more impurities are not bound. The conditions aremodified and the previously bound SIgA antibody is eluted from thecolumn. The opposite can also be performed with the desired antibodypassing though and the impurity or impurities being bound to the column.The light chain constant region can be the affinity target. Usefulaffinity columns include KappaSelect and Capto L from GE Healthcare LifeSciences (Piscataway, N.J., USA). When KappaSelect is used, the additionof MgCl2 is often advantageous. The use of Protein A as an AC column isusually avoided. Another kind of AC step is known as IMAC (immobilizedcopper affinity chromatography). IMAC can be used as the sole AC step orin combination with more traditional AC steps. When used, IMAC is oftencarried out first. If the crude antibody composition, such as theclarified material, contains EDTA, then it is advantageous to add CuSO4to the column in order to remove EDTA, which interferes which the IMACpurification process. Often IMAC is used for small to medium scalepurification of SIgA where the amounts are less than 10 g, typicallyless than 5 grams.

Methods of using SEC for purification of monoclonal antibodies are knownin the art. In general, SEC allows the separation of fully assembledSIgAs of interest from lower molecular forms (such a monomer of IgA,J-chain and SC-chain, or combinations thereof). Furthermore, SEC alsopermits a buffer change, such as, for example, the reformulation of theSIgA of interest into a new desired buffer. Suitable columns include,for example, a Sephacryl S300 HR column.

Other purification steps can be employed as well. For example, ionexchange chromatography (IEX) can be useful for removing coloredimpurities associated with the plant material. Methods and techniquesfor performing IEX chromatographic purification of antibodies are knownin the art and are described, e.g., in Graf et al. (1994) “Ion exchangeresins for the purification of monoclonal antibodies from animal cellculture” Bioseparation, vol. 4, no. 1 pages 7-20, or in “Process scalepurification of antibodies (2009) Edited by U. Gottschalk, ed. J. Wileyand son, Hoboken, N.J., and references cited therein. Often IEX, such asanion exchange chromatography (AEX) or cation exchange chromatography(CEX), is performed before IMAC or other AC step is employed, but is notlimited thereto and can be employed at other points of the purificationand/or can be employed multiple times with the same or differentexchange resin (e.g., AEX and subsequently CEX). In some embodiments anAEX column such as DOWEX 1X2 is employed, often before the AC column.

Further polishing/purification steps can be added, as is known in theart. For example, after any and/or each purification step(chromatography step) an ultrafiltration (UF) step can be performed.Typically, a UF step is performed at or near the end of the polishingphase in order to increase purity and/or change the buffer orconcentration of antibody in the buffer.

The SIgA antibodies are often sufficiently recovered so as to be“isolated” or in an isolated form. Isolation is thus related topurification and is generally achieved by completion of therecovery/extraction step, clarification, and/or capture steps describedabove.

Pharmaceutical Compositions

The SIgA antibodies of the invention can be used in variouspharmaceutical compositions. Typically the pharmaceutical compositioncomprises the antibody and at least one pharmaceutically acceptableexcipient. The pharmaceutical compositions can be solid, semi-solid, orliquid. Generally the pharmaceutical composition is adapted for aparticular route of administration. For example, the pharmaceuticalcomposition can be adapted for oral administration, rectaladministration, buccal administration, topical administration, etc.Preferably, the pharmaceutical composition is adapted for oraladministration.

Pharmaceutical compositions for administering SIgA antibodies viatopical administration include powders, creams, ointments, gels,lotions, solutions and suspensions (including mouth washes). Theexcipient carrier is often aqueous, oil, or polymer based, eachoptionally in the form of an emulsion or microemulsion. The term“topical administration” includes, for example, optical administration(e.g., via a cream/ointment) and administration to the skin (e.g., at aninflamed joint).

Pharmaceutical compositions for administering the antibody via oraladministration include solid oral dosage forms such as tablets,capsules, enteric coated forms thereof, lozenges, and films, as well asliquid dosage forms including solutions, suspensions, liquid filledcapsules, and mouth washes. Tablets can be soluble tablets, dispersibletablets, effervescent tablets, chewable tablets, lyophilized tablets,coated tablets (e.g., sugar-coated or enteric-coated), and modifiedrelease tablets. Capsules include hard gelatin capsules that can befilled with powder, pellets, granules, small tablets, or mini-tablets,or solutions or emulsions or combinations and can be coated for entericor modified release. Soft capsules are also contemplated and are moretypically filled with liquids, gels or dispersions, but are not limitedthereto. Granules can be effervescent granules, coated granules (e.g.,sugar-coated or enteric-coated), and modified release granules. Althoughthe SIgA antibody of the present invention is preferably administeredorally, it should be understood that such administration may beconsidered to be a topical administration to the GI tract. Likewise, asuppository or rectal injection may also be used to topically trat theintestines. The use of an oral dosage form to treat gastrointestinaldisease(s) using the sIgA of the present invention is a specific aspectof the present invention.

Pharmaceutical compositions for administering the SIgA antibody viaparenteral administration are typically liquid. Water is commonly usedas a main excipient, although other pharmaceutically-acceptable liquidssuch as ethanol, glycerol, ethyl oleate, Myglyol, benzyol oleate, castoroil, MCT, benzyl alcohol isopropyl myristate can be used alone or incombination with water or each other. Aqueous compositions that containno other excipients are also contemplated, and can be prepared fromlyophilized, amorphous, or crystalline compounds. Often the injectablecomposition, which can be for subcutaneous, IM, or IV injection,contains isotonizing agents. An injectable solution or suspension istypically sterile, as are all liquid pharmaceutical dosage forms.

An overview of dosage forms can be found in Ansel's PharmaceuticalDosage forms and Drug Delivery Systems. 9^(th) ed. L. V. Allan, N. G.Popovitch, H. C. Ansel, 2010 Lippincott, ISBN: 978-0781779340;Formularium der Nederlandse Apothekers. 2004 WINAp ISBN 90-70605-75-9;Recepteerkunde, G. K. Bolhuis, Y. Bouwman-Boer, F. Kadir en J. Zuiderma,2005 WINAp ISBN 90-70605-65-1; and Apothekenrezeptur und-defektur.Deutscher Apotheker Verlag Stuttgart 1986 ISBN 3-7692-1092-1. See alsoU.S. Pat. No. 7,147,854 for a description of topical preparations fordelivering IL-8 antibodies to treat skin inflammatory disease such aspsoriasis.

The pharmaceutical composition generally contains about 0.01 to 1000 mgof the antibody per dose, depending in part upon the dosage formemployed. The dose can be, for example, fixed or variable (e.g, based onbody weight) Pharmaceutically-acceptable excipients are known in the artand include diluents, carriers, fillers, binders, lubricants,disintegrants, glidants, colorants, pigments, taste masking agents,sweeteners, plasticizers, and any acceptable auxiliary substances suchas absorption enhancers, penetration enhancers, surfactants,co-surfactants, preservatives, anti-oxidants and specialized oils.Specific to the field of biopharmaceutical proteins are excipientsintended to stabilize proteins and cryo-protectants to provideprotection during freeze-drying. Suitable excipient(s) are selectedbased in part on the dosage form, the intended mode of administration,the intended release rate, and manufacturing reliability. Non-limitingexamples of commonly used excipients include polymers, waxes, calciumphosphates, sugars (e.g., trehalose, sucrose, or mannitol), buffers(such as phosphate, acetate, citrate, histidine, or glycine basedbuffers at pH between 5 and 7.5), salts (e.g., NaCl or NaEDTA),polysorbate 20, polysorbate 80, human albumin, dextran, and benzylalcohol.

Treatments

As used herein, the term “treat” or “treatment” means the application oradministration of a SIgA antibody of the invention, alone or as part ofa composition, to a patient with the purpose to cure, heal, alleviate,improve or prevent an inflammatory disease. The term “inflammatorydisease” means a condition associated with symptoms of inflammation,which may be caused by external factors, such as infectious disease, orby internal dysfunctions, such as an autoimmune disease. In thiscontext, the terms disease, disorder, syndrome, condition, and the likeare used interchangeably. In embodiments, the SIgA antibodies of thepresent invention are useful in the topical treatment of inflammatorydiseases in humans, e.g., local administration to the site ofinflammation, such as orally or rectally. Preferably, the SIgAantibodies of the present invention are useful in the oral treatment ofinflammatory diseases.

As used herein, an amount of the SIgA of the present invention effectiveto treat an inflammatory disease, or a “therapeutically effectiveamount,” refers to an amount of the antibody which is effective beyondthat which is expected in the absence of such treatment.

As used herein, the term “patient” is intended to include humans andnon-human animals. The term “non-human animals” includes allvertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles)and mammals, such as non-human primates, sheep, dog, cow, horse, pig,etc. In a preferred instance, the patient is human.

The SIgA antibodies of the present invention are generally useful intreating inflammatory diseases in humans. Specific targets includerheumatoid arthritis, inflammatory bowel disease (including Crohn'sdisease and ulcerative colitis), psoriasis, psoriatic arthritis,ankylosing spondylitis, juvenile idiopathic arthritis, uveitis, asthma,Alzheimer's disease, multiple sclerosis, type II diabetes, systemicsclerosis, lupus nephritis, and allergic rhinitis. The present inventionwill be further illustrated in the following non-limiting examples.

ANTI-TNF-α SIGA EXAMPLES Example 1 Transient Expression of an Anti-TNF-αSecretory Protein Based on Infliximab (IIB-SA2n) in Plants

a) Preparation of cDNA Constructs

The amino acid sequences of a suitable leader sequence (e.g., secretionsignal, SEQ ID NO:23), the heavy chain variable region of infliximab(SEQ ID NO:2), and the human α2(n) IgA heavy chain constant region(Chintalacharuvu et al., Journal of Immunology, 1994, 152, 5299-5304;SEQ ID NO:13) were joined together. Cleavage of the signal sequence(a.a. 1-19) corresponded to the predicted cleavage site using theSignalP program (http://www.cbs.dtu.dk/services/SignalP).

The resulting amino acid sequence was back-translated into a cDNAsequence optimized for expression in maize (Z. mays) (SEQ ID NO:27) (seeLiangjiang Wang and Marilyn J. Roossinck., “Comparative analysis ofexpressed sequences reveals a conserved pattern of optimal codon usagein plants.” Plant Mol Biol (2006) 61:699-710).

Similarly the cDNA sequence for the light chain of the construct wasobtained by joining the sequences of a suitable secretion signal (SEQ IDNO:24), the light chain variable region of infliximab (SEQ ID NO:3), andthe human κ Ig light chain constant region (SEQ ID NO:14), andback-translating the obtained amino acid sequence into a cDNA sequenceoptimized for expression in maize (Zea mays) (SEQ ID NO:29).

The cDNA sequence for the human J-chain was obtained by joining theamino acid sequences of secretion signal (SEQ ID NO:22) and J-chainsequence (SEQ ID NO:20), both obtained from UniprotKB/Swiss-Protdatabase entry P01591, and back-translating the obtained amino acidsequence into a cDNA sequence optimized for expression in maize (Z.mays) (SEQ ID NO:33).

The cDNA sequence for the SC-chain was obtained by joining the aminoacid sequences of a suitable secretion signal (SEQ IDNO:25) and SC-chainsequence (SEQ ID NO:21, amino acids 19-603 of UniprotKB/Swiss-Protdatabase entry P01833), and back-translating the obtained amino acidsequence into a cDNA sequence optimized for expression in maize (Z.mays) (SEQ ID NO:35). The used secretion signal sequence was derivedfrom the natural SC secretion signal by the addition of codons for twoextra amino acids in order to obtain more favorable splicing sites forthe construction of plasmid vectors.

The cDNA's of the four constructs (the heavy chain, the light chain, theJ-Chain and the SC-Chain) were obtained from a commercial source.

b) Vector Construction and Cloning Strategy

The cDNAs for the heavy chain of the construct (HC) and of the J-chain(JC) were ligated into the pGA15 and pGA14 plasmid vectors,respectively, using Pacl and Ascl restriction sites. After transfer toE. coli K12 XL10 gold and expansion, the constructs were eachtransferred to separate pRAP plasmids using NcoI and KpnI restrictionsites, resulting in the expression cassettes 35S:HC:Tnos and35S:JC:Tnos. The pRAP plasmids containing the expression cassettes weretransferred to and expanded in E. coli K12 DH10B.

The cDNAs for the light chain (LC) and of the SC-chain (SC) were ligatedinto the pGA14 and pGA15 plasmid vectors, respectively, using Pad andAscI restriction sites. After transfer to E. coli K12 XL10 gold andexpansion, the constructs were each transferred into separate pTR2plasmids using NcoI and XbaI restriction sites, resulting in theexpression cassettes TR1′TR2′:LC:T35S and TR1′TR2′:SC:T35S. The pTR2plasmids containing the expression cassettes were transferred to andexpanded in E. coli K12 DH10B.

The light chain expression cassette TR1′TR2′:LC:T35S was transferred tothe pRAP vector containing the heavy chain expression cassette35S:HC:Tnos using HindIII restriction sites, and transferred to andexpanded in E. coli K12 DH10B. Finally, the combined cassettescontaining HC and LC (35S:HC:Tnos: TR1′TR2′:LC:T35S) were transferred toa pBIN+ expression vector using AscI and Pad restriction sites. ThispBIN+ vector containing the combined HC and LC cassettes was transferredto Agrobacterium tumefaciens strain MOG101 using electroporation.Similarly the cassettes containing JC and SC (35S:JC:Tnos:TR1′TR2′:SC:T35S) were combined in a pBIN+ expression vector. This pBIN+vector containing the combined JC and SC cassettes was transferred to A.tumefaciens strain MOG101 using electroporation.

The two transfected A. tumefaciens strains were used in combination fortransient plant transformation and expression of the full SIgAconstruct. For vector information see also van Engelen et al., PlantMolecular Biology 1994, 26: 1701-1710. Information on AlMV leadersequence: van der Vossen et al, Nucleic Acids Research 1993, 21:1361-1367. pBIN+ is described in: van Engelen et al., TransgenicResearch 1995, 4: 288-290. (In the referenced literature sources:pRAP35=pCPO31). By way of example, FIG. 6 shows a schematicrepresentation of the building of the pBIN+ vector containing thecombined HC and LC cassettes.

c) Transient Expression in Tobacco Plants

The youngest fully expanded leaves of six week old tobacco plants wereinfiltrated with a mixture of the two A. tumefaciens strains by placinga 2-ml syringe (without needle) containing the bacterial cell suspensionat the lower side of a leaf and gently pressing the suspension into theleaf. The infiltrated area was usually clearly visible. Expression took4-6 days with optimum levels at days 5-6.

The leaves were frozen at −80° C., crushed and 2 ml extraction bufferper gram of leaf material was added. The extraction buffer was PBSpH=7.4; 0.02% Tween-20 (v/v); 2% polyclar AT (v/v); and 1% inhibitorcocktail (v/v). The suspension was homogenized with an Ultra Turrax(Janke & Kunkel). Solid material was removed by centrifugation (15minutes, 10.000 g). The solid material was extracted twice more withextraction buffer, the same as above except the inhibitor cocktail wasreplaced with 10 mM PMSF. The combined extracts were stored at −20° C.Using SDS-page and immunoblotting, formation of a complete SIgA could beshown, but the product was not isolated or purified. The SIgA comprisesIgA(2)(n) heavy chain constant region, human κ light chain constantregion, and the heavy and light chain variable regions of infliximab,and is referred to herein as “IIB-SA2n”

d) Transient Expression in Lettuce

The SIgA construct was transiently expressed in lettuce by vacuuminfiltration using the same vectors as for expression in tobacco (see:Negrouk et al., Highly efficient transient expression of functionalrecombinant antibodies in lettuce., Plant Science 2005, 169, 433-438).Full grown crops of lettuce Lactuca sativa L. (oak leaf lettuce) andvacuum infiltrated with a mixture of the two A. tumefaciens strains andharvested 3-5 days after infiltration. Formation of complete SIgA couldbe shown, but the product was not isolated or purified.

Example 2 Expression of an Anti-TNF-α Secretory Protein Based onInfliximab (JIB-SA2n) in Mammalian Cells 2a: Transient Expression ofIIB-SA2n

(1) Vector Generation:

cDNAs encoding SIgA heavy chain (HC), light chain (LC), J-chain, andSC-chain were cut from the pGA14 and pGA15 plasmids described inExample 1. The excised HC and LC sequences were PCR amplified and clonedinto pMQR-hIgG1 (Neo) to give pMQR-zmα2-iib (Neo) and pMQR-zmκ-iib (Neo)vectors. Similarly, the excised J-chain and SC-chain were PCR amplifiedand cloned into pMQR-kappa (Hygro) to give the pMQR-zmJ (Hygro) andpMQR-zmSC (Hygro) vectors. The pMQR-hIgG1 (Neo) and pMQR-kappa (Hygro)vectors are based on the pcDNA3 (Invitrogen) backbone, which wasmodified to receive any combination of Ig V- and C-region “cassettes”.This was performed by subcloning well-defined genomic Ig gene segmentsinto pcDNA3, after which silent mutations were introduced flanking theV- and C-regions to create restrictions sites allowing for the exchangeof V- and C-gene segments. Other occurrences of these specificintroduced restriction sites were removed from the vector backbone toensure proper exchange of V and C regions on unique restriction sites.In short, the BsmI site was removed by exchanging the original BsmIsequence (5′-TGCATTC-3′) present in the vector into 5′-TGCAAAC-3′.Furthermore the MunI/MfeI and HindIII sites were destroyed byrestriction digestion followed by end-filling and re-ligation.

Large-scale production of each of the four expression vectors fortransfection was performed using Maxiprep-kits (Sigma).

(2) Cell Transfection and Culture:

Transient SIgA producing CHO-S and HEK-293F cells (Invitrogen) andHEK293-EBNA1 (ATCC CRL-10852) were made as follows: Invitrogen derivedcell types were used as host for transfection using FreeStyle Max orAmaxa Nucleofection. HEK293-EBNA1 was used for large scale (10 l)transient polymer transfection. To obtain a complete SIgA-producingtransfectant, all 4 different vectors were transferred simultaneouslyinto each of these cell types (CHO-S, HEK293F, and HEK203-EBNA1).

(3) FreeStyle MAX Transfection

To transfect 30×10⁶ CHO-S and HEK-293F cells (separately), 9,375 ug ofeach vector was used. SIgA production was performed by culturing cellsin a 200 ml shake flask at 37° C., 130 rpm and 8% CO₂ during 120 hoursin respectively CHO-S FreeStyle and 293F FreeStyle medium. The cellconditions and transfection efficiency were determined by GUAVA ViaCountand Expresse Pro analysis to guarantee the optimal cell growthconditions in between 0.5×10⁶ to 2.0×10⁶ cells/ml. Each time the cellshad to be diluted the volume was increased and if necessary transferredto a 500 ml baffled shake flask.

CHO-S/SIgA 24 hours post transfection: Transfection efficiency; 25.04%and cell viability; 91.59%.

HEK-293F/SIgA 24 hours post transfection: Transfection efficiency;25.52% and cell viability; 80.36%.

(4) Amaxa Transfection

To transfect 1×10⁶ Invitrogen's CHO-S or HEK-293F cells 1 ug of eachvector was used. SIgA production was performed by static culturing cellsin a T25 flask at 37° C. and 5% CO₂ during 96 hours. At 96 hours posttransfection cell suspensions fractions were taken for productionanalysis.

CHO-S/SIgA 24 hours post transfection: Transfection efficiency; 54.27%and cell viability; 94.19%.

HEK-293F/SIgA 24 hours post transfection: Transfection efficiency;61.57% and cell viability; 68.15%.

(5) Polymer Transfections

Transfections of HEK293-EBNA1 cells were performed essentially asdescribed by Durocher at al. Nucleic Acids Res. 30, E9 (2002) (see alsoMorlot C. et al., Acta Crystallogr Sect F Struct Biol Cryst Commun. 63,689-91 (2007)). The day prior to transfection, the cells were routinelydiluted 4-5 times in fresh Freestyle medium (without further addition ofFCS or G418) to a density of 0,3×10⁶/ml. The next day transfectionmixtures were prepared. For transfection of 1 litre of cell culture 500μg of high quality DNA was diluted in 25 ml OptiMEM medium (Invitrogen)1 ml of a 1 mg/ml stock solution of linear 25 kDa PEI (Polysciences) wasadded to the mixture and immediately vortexed for 10 seconds. After 10minutes for incubation the DNA:PEI mixture was added to the cells. Cellsor conditioned medium were routinely harvested six days posttransfection. Culture was performed at 10 L scale. The anti-TNF-αsecretory IgA antibody, IIB-SA2n, in the supernatant of this culture canbe isolated and purified as described in Example 3a.

2B: Stable Expression in Hek293F of IIb-Sa1

(1) Vector Construction

cDNA encoding the heavy chain variable region of infliximab was cut fromthe previously obtained vector pMQR-zmα2-iib (Neo) (see Example 2a(1)).This cDNA was fused into a pMQR vector containing the human genomic DNAsequence encoding the IgA α1 heavy chain constant region to give vectorpMQR-huα1-iib (Neo). Similarly, the light chain variable region ofinfliximab was cut from pMQR-zmκ-iib (Neo) and transferred to a pMQRvector containing the human genomic DNA sequence encoding the κ-lightchain constant region to give vector pMQR-huκ-iib (Neo). The two vectorspMQR-huα1-iib (Neo) and pMQR-huκ-iib (Neo) were fused into a singlevector pMQR-huIgA1-iib (Neo) (SEQ ID NO:28).

Previously obtained vectors pMQR-zmJ (Hygro) and pMQR-zmSC (Hygro) werealso combined into a single vector PMQR-zmJ/SC (Hygro) (SEQ ID NO:30).

(2) Transfection

HEK-293F cells were transformed with both of the two vectors describedabove using polymer transfection as described in experiment 2a(5). Cellswere grown in neomycin and hygromycin containing medium. After recovery,cells were grown on semisolid medium with neo/hygromycin and clones wereselected using Clonepix analysis and selection. A suitable clone (2D5)was selected. Clone 2D5 was expanded under various conditions andscales. Ultimately 15 L of culture medium containing 440 mg of completeSIgA (according to ELISA analysis) was obtained from the expanded 2D5cells. The SIgA comprises IgA(1) heavy chain constant region, human κlight chain constant region, and the heavy and light chain variableregions of infliximab; and is referred to herein as “IIB-SA1.” IIB-SA1can be isolated from culture medium and purified as described in Example3b.

Example 3 Purification of Anti-TNF-α Secretory IgA Antibodies (IIB-SA2nand IIB-SA1) from Mammalian Cell Culture

3a—Affinity and SEC (IIB-SA2n)

The supernatant obtained from the transient expression of IIB-SA2n inHEK-293-EBNA1 cells using polymer transfection (experiment 2a(5)) waspurified using affinity chromatography with an anti-IgA lama antibodyfragment immobilized on sepharose (CaptureSelect human IgA, BAC, productcode 2880) followed by SEC.

(1) Affinity Chromatography:

Anti-human IgA matrix (Capture Select) was poured into a XK16/20 column(16×200 mm) in 20% ethanol in MilliQ water at a flow rate of 15 ml/min.After adjusting the position of the upper flow adaptor, the column wasequilibrated in PBS buffer pH 7.4. The HEK-cell culture medium wasfiltered to remove large cell debris and subsequently loaded on thepre-equilibrated anti-human IgA column with a flow rate of 4.5 ml/minAfter washing with 10 column volumes PBS pH 7.4, the active secretoryIgA antibody was isocratically eluted from the column with 5 columnvolumes of citrate/arginine/NaCl buffer pH 2.5, followed by immediateneutralization to pH 7.7 with a 1 M Tris stock solution.

(2) SEC Chromatography:

In the second step, the adjusted pool from the first step wasconcentrated 4-fold down to 5 ml using a Vivaspin 15R device (30 kDacut-off filter) and loaded on a prepacked Superdex 200 16/60 GL columnequilibrated with PBS pH 7.4. The sample loading volume was 4% of thebed volume and the flow rate was 1.5 ml/min. This size exclusion gelfiltration step resulted in fractionation of SIgA and monomeric IgAaccording to their molecular weights. The elution fractions containingSIgA or monomeric IgA as determined by non-reducing SDS-PAGE were pooledseparately and subsequently concentrated 10-fold using a Vivaspin 15Rdevice (30 kDa cut-off filter).

Samples of substantially pure IIB-SA2n and the corresponding monomericanti-TNF-α IgA were collected and characterized.

3b—Jacalin Affinity and SEC (JIB-SA1)

Culture medium obtained from HEK-293F clonal cell line 2D5 expressingIIB-SA1 (see Example 2b) was purified using Jacalin affinitychromatography followed by SEC.

(1) Jacalin Affinity Chromatography

500 ml culture medium was filtered through a 0.45/0.2 μm Sartopore 2filter. A Jacalin column was prepared by packing 15 ml of immobilizedJacalin resin (Pierce art nr. 20395) in an omnifit 15 mm glass column.The column was equilibrated using 5 column volumes of PBS buffer pH 7.4.Culture medium was loaded at a flow rate of 3 ml/min. after washing with5 column volumes of PBS buffer pH7.4 at 5 ml/min followed by elution ofbound material with 8 column volumes of PBS buffer pH7.4 containing 0.5Mgalactose. The fractions of the eluate containing SIgA were pooled(detection by UV OD280 measurement). The pooled eluate (64 ml) wasconcentrated to a volume of 5 ml using Vivaspin filters. Measurement ofSIgA content using ELISA showed that only appr. 30% was bound to theJacalin, the remainder being in the flow-through fraction.

(2) SEC Chromatography

The concentrated eluate was placed in a superloop and loaded on aSephacryl S300 column (GE-Healthcare art.nr. 17-1167-01) with a flowrate of 1 ml/min. The product was eluted using 1.5 column volumes of PBSbuffer (50 mM phosphate pH 7.4) containing 0.5M arginine. Thechromatogram (UV OD280 detection) showed three overlapping peaks.Fractions were analysed by SDS-PAGE to identify the SIgA containingfraction. The fractions containing the product were pooled. Reducing andnon-reducing SDS-PAGE analyses of the purified product, IIB-SA1, areshown in FIG. 7.

Example 4 Stable Expression of Anti-TNF-α Secretory IgA Antibody(ADB-SA1g) in Lemna

Anti-TNF-α SIgA was produced in Lemna by stable insertion of DNA codingfor the following proteins: The amino acid sequence of the heavy chainconsisted of the Oryza sativa (rice) amylase secretion signal (SEQ IDNO:26) attached to the N-terminal amino acid of the heavy chain variableregion of adalimumab (anti-TNF-α IgG1, Humira®, CAS number 331731-18-1)(SEQ ID NO:4) attached to the N-terminal amino acid of a human IgA1heavy chain constant region (SEQ ID NO:10). The amino acid sequence ofthe light chain of the Anti-TNF-α SIgA combines the rice α-amylasesecretion signal (SEQ ID NO:26) with the TNF-α binding light chainvariable region of adalimumab (SEQ ID NO:5) and the human κ-light chainconstant region (SEQ ID NO:14). The amino acid sequence of the J-chainconsisted of the rice α-amylase secretion signal (SEQ ID NO:26) attachedto the N-terminal amino acid of the human J-chain (SEQ ID NO:20). Theamino acid sequence of the SC-chain consisted of the rice amylasesecretion signal (SEQ ID NO:26) attached to the N-terminal amino acid ofthe human SC-chain (SEQ ID NO:21). The produced SIgA, having IgA1 heavychain constant region, human κ-light chain constant region, andadalimumab heavy and light chain variable regions, combined with humanJ-chain and SC-chain is referred to as ADB-SA1.

Genes were designed for each of the four components with Lemna minorpreferred codon usage (63-67% GC content). Tables with suitablepreferred codon use in Lemnaceae can be found in PCT applicationWO2005/035768 and in relevant references contained therein. Thesynthetic genes also contained the rice α-amylase signal sequence(GenBank M24286; SEQ ID NO:26) fused to the 5′ end of their codingsequences. Restriction endonuclease sites were added to allow cloninginto A. tumefaciens binary vectors. Design of DNA sequences and vectorconstruction was performed by Biolex Therapeutics, Inc., Pittsboro,N.C., USA. DNA sequences were produced by DNA2.0 (Menlo Park, Calif.,USA).

The ADB-SA1g antibody was expressed in L. minor by transfection via anA. tumefaciens binary vector containing DNA sequences encoding all fourof the SIgA components: J-chain (SEQ ID NO:34), SC-chain (SEQ ID NO:36),H-chain (SEQ ID NO:31) and L-chain (SEQ ID NO:32). To prepare thisvector, independent expression cassettes were created containing apromoter and also DNA sequences encoding the protein and terminator forthe J-chain, SC-chain, H-chain and L-chain. The H chain was fused to themodified chimeric octopine and mannopine synthase promoter with Lemnagibba 5′ leader from ribulose bis-phosphatecarboxylase small subunit-1.The L-chain, SC-chain and J-chain genes were fused to high expressionLemnaceae Ubiquitin promoters L. minor polyubiquitin promoter (LmUbq),Lemna aequinoctialis polyubiquitin promoter (LaUbq) and Spirodelapolyrrhiza polyubiquitin promoter (SpUbq), respectively. Sequences ofthese promoters have been disclosed in PCT application WO2007/124186.These expression cassettes were then cloned into a single A. tumefacienstransformation vector EC2.2 (a modification of the A. tumefaciens binaryvector pBMSP3, which is a derivative of pBINPLUS. See Ni, M., Cui, D.,Einstein, J., Narasimhulu, S., Vergara, C. E., and Gelvin, S. B., PlantJ. 7, 661-676, (1995), van Engelen, Transgenic Res. 4:288-290 (1995),and Gasdaska et al., Bioprocessing J., 3:50-56 (2003)), with theappropriate restriction sites to create the final transformation vectorSynB02. This vector also contained the gentamicin acetyltransferase-3-Igene (aacC1) which confers resistance to gentamicin and allows forselection of transgenic L. minor lines.

SynB02 was used to create an additional transformation vector togenerate a glycan optimized version of anti-TNF-α SIgA, having G0glycans lacking fucose and xylose residues. A chimeric hairpin RNA wasused to silence endogenous L. minor mRNAs encodingα-1,3-fucosyltransferase (Fuct1, GenBank DQ789145) andβ-1,2-xylosyltransferase (Xylt1, GenBank DQ789146). A DNA sequence forthis chimeric RNAi molecule was fused to the high expression SpUbqpromoter and subsequently moved into the SynB02 vector creating thefinal transformation vector SynB03. Further details on production ofglycan optimized proteins in Lemnaceae can be found in PCT applicationsWO2007/084672, WO2007/084922, WO2007/084926 and in Cox, K. M., NatureBiotechnology 2006, 12: 1591-1597.

L. minor strain 8627 was transfected with vector SynB03, andglycosylation modified L. minor strain XF04 was transfected with vectorSynB02. Once transformed plants were regenerated (approximately threemonths), single plants were harvested from the antibiotic selectionplates and propagated separately in liquid growth media, withoutselection antibiotic, for further screening and characterization. Thusseveral hundred individual transgenic plant lines from each constructwere generated. Independent transgenic lines were harvested and clonallypropagated in individual harvest jars. For screening of transgeniclines, clonal lines were preconditioned for 1 week at light levels of150 to 200 μmol/m²·s in vented plant growth vessels containing SH medium(Schenk R. U. et al., Can. J. Biol. 1972, 50: 199-204) without sucrose.Fifteen to twenty preconditioned fronds were then placed into ventedcontainers containing fresh SH medium, and allowed to grow for twoweeks. Tissue samples from each line were collected and frozen foranalysis.

To determine SIgA expression, frozen tissue samples were homogenized andcentrifuged, and the supernatant was removed and screened by an ELISAmethod using sheep anti-human IgA secretory chain (AbD Serotec catalog#5111-4804-1:1000 dilution) coated plates to capture the SIgA antibody.The samples were then detected using a goat anti-human kappa light chainHRP conjugated antibody (Sigma catalog #A7164-1:2000 dilution). Thehighest-expressing lines from this primary screening were then grownagain for two weeks in small research vessels under the optimal growthconditions. The resulting tissue was harvested and the ELISA wasperformed to determine the percent of the total soluble protein that isthe expressed SIgA antibody (ADB-SA1g). The results are summarized inTable 3.

TABLE 3 Hightest Construct Glycosylation # of lines screened Expressionlevel SynB02 G0 glycans 55 16.2% TSP SynB03 G0 glycans 227 13.6% TSP

Results of non-reducing and reducing SDS-PAGE analyses of purifiedmaterial obtained from Lemna transfected with construct SynB02 are shownin FIG. 8. ADB-SA1g can be isolated from Lemna culture and purified asdescribed in Example 5.

Example 5 Isolation and Purification of Anti-TNF-α Secretory IgAAntibody ADB-SA1g from Lemna

Biomass from transgenic Lemna expressing anti TNF-α SIgA, havingvariable regions that are the amino acid sequence of the variableregions (antigen binding regions) of adalimumab, was homogenized in 50mM Sodium phosphate, 0.3M Sodium chloride, buffer pH 7.4, at a buffer totissue ratio of 4:1. An acid precipitation step was performed on thecrude extract to remove ribuloase bis-phosphate carboxylase (RuBisCo)and other plant proteins by adjusting the extract to pH 4.5 using 1MSodium acetate, pH 2.5. The precipitate was removed by centrifugation ofthe material at 14,000×g for 30 minutes at 4° C. The supernatant wasadjusted to pH 7.4 and loaded on DOWEX (Dowex 1X2 anion exchange resin)to remove colored impurities. The flow-through fraction containing antiTNF-α SIgA was 0.22 μm was filtered prior to chromatography usingaffinity chromatography and Size Exclusion Chromatography.

Affinity purification: A KappaSelect (GE Healthcare prod. Nr.17-5458-03) column was prepared according to manufacturer instructions(28-9448-22 AA). The column was equilibrated with 5 column volumes (cv)of TBS buffer (50 mM Tris, 0.15M Sodium chloride, pH 7.4). Thesupernatant was loaded on the column Approximately 5 mg sIgA/ml resinwas loaded on columns of up to 1 L KappaSelect. Non-binding material waswashed from the column with 5 cv TBS buffer. The product was eluted fromthe column using 10 cv of 25 mM Sodium Acetate, pH 6.6 buffer containing3.5 M MgCl₂. The fractions containing the secretory proteins were pooledand immediately diluted 4-fold using TBS buffer (50 mM Tris, 0.15MSodium chloride, pH 7.4). MgCl₂ was replaced by buffer exchange with atleast 10 volumes of TBS buffer using a Pall Centramate cassetteultrafiltration system and a Pellicon 2 Mini Filter (Millipore prod.Nr.P2C005C01-PLCCC 5K, regenerated cellulose) with 5 kDa cut-off.

SEC purification: Material obtained by KappaSelect chromatography wasfurther purified on a Sephacryl 5300 HR column. The column wasequilibrated with 2 cv of PBS buffer at a flow rate of 1.0 ml/min. TheKappaSelect eluate was concentrated 3-4× using ultra filtration using aPellicon 2 Mini Filter (Millipore prod.Nr. P2C005C01-PLCCC 5K,regenerated cellulose) with 5 kDa cut-off.

Typically, a concentration of 3-5 mg/ml and feed volume of 50 ml wasused for a 1 L column. The feed was applied using an AKTA purifier at1.0 ml/min. Elution was performed with at least 2 column volumes of PBSbuffer at room temperature and a flow rate of 1.0 ml/min Fractions werecollected and sufficiently pure fractions were pooled.

Example 6 Binding of Anti-TNF-α SIgA IIB-SA1 to TNF-α

Binding of IIB-SA1 (infliximab binding head) after purification asdescribed in Example 3b, to TNF-α was determined using ELISA accordingto the following procedure.

96-Well plates were incubated overnight at 4° C. with 100 μl/well of 0.5μg/ml TNF-α in PBS pH7.4. After emptying the wells were incubated for 2hours at RT with 200 μl/well of PBS pH7.4 containing 1% BSA and 0.05%Tween-20. After washing (room temperature) with 300 μl/well of PBS/0.05%Tween-20 three times for 30 seconds, shaking, the plates were used. Thewells were incubated with samples, 1 hour at 37° C., 100 μl/well. Thewells were washed (room temperature) with PBS/0.05% Tween-20 three timesfor 30 seconds, shaking, 300 μl/well. The wells were incubated with 100μl/well of 0.5 μg/ml HRP labeled anti-human kappa light chain antibody(Sigma, A7164) for 1 h at RT, and then washed (room temperature) withPBS/0.05% Tween-20 three times for 30 seconds, shaking, 300 μl/well.

For detection, the wells were incubated with TMB (Tebu Bio), 10 minutesat room temperature, 100 μl/well. The reaction was stopped with 0.3 Msulphuric acid, 100 μl/well, and the OD at 450 nm was measured with wellcorrection at 630 nm. Measurement of IIB-SA1 from expression inmammalian cells (Example 2b) showed strong TNF-α affinity at a levelcomparable to that of infliximab as shown in FIG. 9. Note that theclosed circle represents the SIgA of the invention (IIB-SA1), squarerepresents Infliximab, and diamond represents Colostral SIgA.Equilibrium constants of 8.1×10⁻¹⁰ and 2.4×10⁻¹⁰ for IIB-SA1 and forInfliximab itself (REMICADE®), respectively, were calculated. This showsthat the transfer of the TNF-α binding variable part of IgG1 antibodyinfliximab to a secretory IgA does not substantially alter affinity(taking the variation and discriminatory power of the experimental setupinto account), and that the obtained product is fully functional.

Example 7 Proteolytic Stability of Anti-TNF-α SIgAs (IIB-SA1 andADB-SA1g)

The stability of IIB-SA1 (Example 3b), and of ADB-SA1g (Example 4, fromSynB03) was determined in simulated intestinal fluid (SIF, 0.05Mphosphate buffer pH 6.8 containing 10 mg/ml pancreatin). Stability wascompared to a human colostral SIgA which was purified to contain onlykappa light chains and α-1 heavy chains.

150 μl of a 0.14 mg/ml solution of the material to be tested (i.e.,IIB-SA1, ADB-SA1g, or human colostral SIgA (kappa light chain, α-1 heavychain) was added to 550 μl of pre-heated SIF at 37° C. 100 μl sampleswere drawn at T=0, 5, 15, 30, 60 and 120 min, added to 25 ul proteaseinhibitor cocktail (Roche, 1169749800) followed by immediate freezing inliquid nitrogen. Samples were analyzed by non-reducing SDS-Page gelelectrophoresis. Briefly; 57.5 μl of a 0.08 M solution of iodoacetamidein LDS sample buffer was added to each of the frozen samples. Sampleswere thawed and applied to Criterion Tris-HCl gel (12.5%, 18 well, 30 μlcomb (Biorad, 345-0015). After electrophoresis gels were treated withKrypton protein stain and analyzed. Stability of the samples wasqualitatively assessed visually. Results are shown in FIG. 10.

The two forms of anti-TNF-α SIgA, i.e., IIB-SA1 (labeled as infliximabvariable part in FIG. 10) and ADB-SA1g (labeled as adalimumab variablepart in FIG. 10) each showed a stability which was slightly less thanthat of the colostral SIgA.

Example 8a Caco-2 Cell Based Assays

The aims of Caco-2 cell experiments were: (a) to evaluate in vitro thecapacity of anti-TNF-α SIgA's to block the biological activity of TNF-αand (b) to study transport over an epithelial cell monolayer.

Caco-2 Cell Culture Conditions and Exposure to TNF-α

Human colonic adenocarcinoma epithelial Caco-2 cells (HTB 37, AmericanType Tissue Collection) were grown in C-DMEM consisting of DMEM-Glutamax(Life Technologies) supplemented with 10% fetal calf serum (FCS), 1% nonessential amino acids, 10 mM HEPES, 0.1% transferrin and 1%streptomycin/penicillin Cells cultivated to 80% confluency were seededon Snapwell filters (diameter, 12 mm; pore size, 0.4 mm; Corning Costar)at a density of 0.4×10⁵ cells/cm². The formation of a polarized Caco-2cell monolayer at week 3 was established by morphology (laser scanningconfocal microscopy) and monitoring of the transepithelial electricalresistance (TER; 300-350Ω×cm²) using a Millicell-ERS apparatus(Millipore). After formation of the Caco-2 cell monolayer, the apicalcompartment medium was replaced by culture medium lacking FCS. One hourlater, the medium lacking FCS was then replaced by medium containing 10or 20 ng/ml of TNF-α, The polarized Caco-2 cell monolayers weresensitized with the TNF-α for 28 hours and the integrity of themonolayer was monitored by measurement of the TER.

Effect of SIgA's on Polarized Caco-2 Cell Monolayer Sensitized by TNF-αApplied Apically

Caco-2 cell monolayers were established as described above. For eachSnapwell insert, 10 ng TNF-α was mixed with an equimolar, or a 10-foldmolar excess, of an antibody IIB-SA1 or ADB-SA1g to a final volume of 40microliters phosphate-buffered saline (PBS). The TNF and antibodymixture was incubated for 20 minutes at ambient temperature followed bydilution with 460 microliters of culture medium lacking FCS. The mediumin the apical compartment was replaced by the TNF and antibody mixture.The Caco-2 cell monolayers were sensitized for 28 hours and theintegrity of the monolayer was monitored by measurement of the TER.

Results:

As shown in FIG. 11 for the 10-fold molar excess assay, taking themedium condition as 100%, the drop in TER values induced by TNF-α couldbe completely compensated by the presence of either anti-TNF-α SIgApreparation. The data are a mean of 6 independent replicates for eachcondition. Statistical analysis using ANOVA followed by Dunnett'sMultiple Comparison test found a p<0.001 for the ADB-SA1g and IIB-SA1results (indicated with *** in FIG. 11).

Effect of SIgA's on Polarized Caco-2 Cell Monolayer Sensitized by TNF-αApplied Apically: Possible Recovery after Apical Delivery.

Polarized Caco-2 cell monolayers were established as described above.The medium in the apical compartment was replaced by 20 ng/ml TNF-α asindicated above, and the polarized Caco-2 cell monolayers weresensitized for respectively 15 or 28 hours prior to addition of SIgA's(specifically IIB-SA1 or ADB-SA1g). After sensitization, Caco-2 cellwere exposed to SIgA's for 24 hours and TER was monitored.

Results:

The results are shown in FIG. 12. Black bars represent exposure to TNF-αonly, striped bars represent addition of ADB-SA1g after indicated timeof exposure to TNF-α, dotted bars represent addition of IIB-SA1 afterindicated time of exposure to TNF-α. Statistical analysis using ANOVAfollowed by Dunnett's Multiple Comparison test, ADB-SA1g and IIB-SA1were compared to TNF-α and had a p<0.001 (indicated by *** in FIG. 12).Data are means of 4 independent replicates Snapwell inserts for eachcondition. As expected, no effect was seen when adding the SIgAcompounds at t=15 h, a time-point when the TER is not yet affected. At alater time-point (28 hours), which is known to dramatically affect TER(see e.g., Dongmei Ye, et al., Am J Physiol Gastrointest Liver Physiol290: pp 496-504, 2006), application of the SIgA compound in the apicalcompartment resulted in marked recovery of the TER measured 24 hourslater.

Conclusion:

These data suggests that delayed apical neutralization of the effect ofTNF-α on polarized Caco-2 cell monolayers can lead to recovery, as longas the SIgA's are delivered within 28 hours post-cytokine exposure.

Tracking of the Bio-Availability of Fluorescent ADB-SA1g Applied in theApical Compartment of Polarized Caco-2 Cell Monolayers

ADB-SA1g was labeled with indocyanine Cy5 using the antibody labelingkit (General Electrics Healthcare), following the procedure provided bythe kit's manufacturer. One microgram of labeled ADB-SA1g diluted in 500microliters of FCS-free Caco-2 cell culture medium was added to theapical compartment and incubated overnight. Snapwell inserts were washedtwice with PBS prior to fixation overnight with 5 ml of 4%paraformaldehyde at 4° C. After washing with PBS, filters werepermeabilized with 0.2% Triton X-100, and non-specific binding siteswere blocked with 5% FCS in PBS. Inserts were incubated with rabbitanti-human zonula occludens-1 (ZO-1) (1/200 dilution), washed in PBSprior to addition of secondary Ab (goat anti-rabbit IgG conjugated withAlexa Fluor 647; 1/100 dilution). After PBS washing, filters were thenincubated with 4′,6-diamidino-2-phenylindole (DAPI) at a concentrationof 100 ng/ml in PBS (Invitrogen) for 30 min. Filters were recovered fromtheir holders, and mounted in Vectashield (Vector Laboratories) forobservation using a Zeiss LSM 710 Meta confocal microscope (Carl Zeiss,Germany) equipped with a 40× objective (Cellular Imaging Facilityplatform, Lausanne University, Switzerland) and processed using ZeissZEN 2009 light software.

Results:

Because 3D image reconstitution does not allow to finely pinpoint thelocation of the SIgA in contact with the Caco-2 cell monolayer, reliabletracking was carried out by analyzing successive confocal plans. Cellsections were selected at the bottom of microvilli, at the level oftight junctions, in the nuclear periphery, and at the level of thenucleus. Proper visualization was ensured by simultaneous staining withanti-ZO-1 antibodies and DAPI. ADB-SA1g was localized on the cellsurface. The antibody was detected in the form of dense fluorescentspots within the cell cytosol, most likely in endocytotic/micropinocyticcompartments.

Conclusion:

This indicates that intracellular distribution may serve as second lineof defense and that ADB-SA1g could eventually neutralize internalizedTNF-α.

Example 8b Localization of Anti-TNF SIgA ADB-SA1 g in In-Vivo AnimalModels of IBD

In an animal model of IBD, disease was induced in female C57Bl/6 mice, 8weeks of age, by topical sensibilization (day 1) with 0.5% TNBS inacetonitril (4:1), followed by a rectal challenge of 2.5% TNBS in 100%Ethanol on day 4 Animals were treated orally 3 times with ADB-SA1g (100μg/mouse): 2 days before topical sensibilization (day −1) and on day 1and 4. At day 6, when disease symptoms became apparent, animals weresacrificed and distal colon was collected for histological analyses.Fresh frozen tissue was fixed in 4% paraformaldehyde and washed 3 timeswith wash buffer (1×PBS+2% BSA). Unspecific binding sites were blockedwith protein block reagent, washed 3 times and incubated with primaryantibody over night at 4° C. After 3 wash steps, tissue slides wereincubated with secondary antibody for 4 hours at 4° C., washed 3 timesand incubated with streptavidin-Alexa488 for 1 hour at 4° C. Finallytissue slides were mounted with Vectashield mounting medium. Primaryantibodies used were: sc-20656 (human SC chain) rabbit (Santa Cruz)1:200, CD31 (Dendritic cells) rat antibody (Biolegend) 1:200, CD326(EpCam) rat antibody (Biolegend) 1:200, CD11c (Dendritic cells)arm.Hamster (Biolegend) 1:200 and M-cells ULEX-1: lectin-FITC directlyconjugated (Sigma-Aldrich) 1: 200. Secondary antibodies used were:donkey-anti-rabbit Dylight 549 nm (red) (Dianova) for sc-20565,goat-anti-rat biotin antibody (BD Pharmingen)1:500+Streptavidin-Alexa488 nm (green) 1:200 for CD31 and CD326, andgoat-anti-arm. Hamster biotin antibody (Caltag) 1:500+StreptavidinAlexa488 nm for CD11c.

Results: To demonstrate the presence of ADB-SA1g, antibody sc-20656 wasused. This antibody binds to the human secretory chain, but not to mousesecretory chain. Positive staining for ADB-SA1g was found in epithelialcells (positive for CD 326) and in dendritic cells (positive for CD11c+and CD31+). Surprisingly, no co-localization of ADB-SA1g with M-cellswas found under the above described experimental conditions.

Conclusion:

This data confirms the “tracking of the bio-availability” results fromExample 8a and indicates that intracellular distribution may serve assecond line of defense and that ADB-SA1g could eventually neutralizeinternalized TNF-α.

Example 9 Efficacy of Anti-TNF SIgA ADB-SA1g in In-Vivo Animal Models ofIBD

C57BL/6 mice were pre-treated orally 5 times a week (not in weekend)with PBS control, ADB-SA1g (100 μg/mouse), or 3 times per weeksubcutaneous with Adalimumab (100 μg/mouse). Start of pre-treatment ist=0. At day t=7 mice were treated with 2% dextran sulfate sodium (DSS)in drinking water to induce inflammation symptoms in intestine. Firstsigns of inflammation can be seen at day 10-12, together with declinedbody weight. With mini-endoscopic system an analysis of the status ofdisease was performed. Briefly, a mini-endoscope (1.9 mm outer diameter)was introduced via the anus and the colon was carefully inflated with anair pump. Endoscopic pictures obtained allow the monitoring and gradingof inflammation. Thereafter, endoscopic scoring of five parameters from0-3 (1; translucent structure, 2; granularity, 3; fibrin, 4;vascularity, and 5; stool) resulting in the overall score from 0 (nochange) to 15 (severe colitis) was performed. Typically, after one weekexposure to DSS the symptoms are so strong that mice need a restingperiod to recover. At day t=16 the experiment was terminated andclinical score analyzed.

The results are depicted in FIG. 13. Statistical analysis using ANOVAfollowed by Dunnett's Multiple Comparison test was performed comparingall groups to PBS control group. The ADB-SA1g was significant having ap<0.001 (indicated as *** in FIG. 13). n=7 mice per group. Oraltreatment with ADB-SA1g significantly inhibited DSS-induced inflammatorybowel disease in C57BL/6 mice. In contrast, subcutaneous treatment withAdalimumab was not effective, even though both antibodies used the samebinding head (variable region).

In FIG. 14 a representative mini-endoscopic picture of the colitis scoreat day t=15 is depicted.

Example 10 Efficacy of Anti-TNF SIgA ADB-SA1g in an In-Vivo Animal Modelof RA

The therapeutic efficacy of oral ADB-SA1g was evaluated in a rheumatoidarthritis (RA) model based on a TNFα-overexpressing transgenic mousemodel which spontaneously develops arthritis.

TNF^(ΔARE/+) male mice (BiomedCode Hellas SA,http://www.biomedcode.com/content.php?page=tnf-dare), heterozygous forthe mouse TNFα mutation (maintained in a C57BL/6J genetic background)were crossed with C57BL/6J females. Their heterozygous offspring used inthis study were identified by tail DNA genotyping which was furtherconfirmed by the phenotype of these animals which exhibit a ruffled furcoat. Mixed sex mice were used in this study.

From the fourth week of age and onwards, the mice received daily(Monday-Friday) by oral gavage either drinking water, buffer (vehicle),ADB-SA1g (5 mg/kg), or subcutaneously every other day (Mon-Wed-Fri)etanercept (10 mg/kg).

Body weight and grip strength measurements were recorded weekly for eachmouse. All TNF^(ΔARE/+) mice showed normal body weight gain whencompared to wild-type littermate control mice. Statistical analysisusing ANOVA followed by Newman-Keuls Multiple Comparison Test wasperformed comparing all groups to TNF^(ΔARE/+) vehicle treated group(n=6-8 per group, ** p<0.01, *** p<0.001). At 11 weeks of age, thevehicle-treated TNF^(ΔARE/+) mice displayed significantly reduced gripstrength in comparison to wt littermate control mice andetanercept-treated TNF^(ΔARE/+) mice. Mice treated orally with ADB-SA1gand mice treated subcutaneously with etanercept displayed comparable,statistically-significant increased grip strength (FIG. 15).

At 11 weeks of age, when the control TNF^(ΔARE/+) group had obvioussigns of arthritic pathology, mice from all groups were sacrificed andtissue samples from ankle joints were collected and processed forhistopathological analysis. Ankle joints were assessedhistopathologically. The hind joints of the TNF^(ΔARE/+) mice werescored for synovial hyperplasia, existence of inflammatory foci,cartilage destruction, and bone erosion using the scoring scale detailedbelow:

-   -   0=normal    -   1=mild inflammation in periarticular tissue and/or mild oedema    -   2=moderate inflammation and pannus formation with superficial        cartilage and bone destruction    -   3=marked inflammation with pannus formation and moderate        cartilage and bone destruction (depth to middle zone)    -   4=severe inflammation with pannus formation and marked cartilage        and bone destruction (depth to tidemark).

Histopathological analysis of the ankle joints revealed that the vehicletreated TNF^(ΔARE/+) mice displayed severe signs of arthritis.Statistical analysis using ANOVA followed by Newman-Keuls MultipleComparison Test was performed comparing all groups to TNF^(ΔARE/+)vehicle treated group (n=6-8 per group, * p<0.05, ** p,0.01).Etanercept-treated animals displayed significantly decreased signs ofpathology. Mice treated orally with ADB-SA1g also showed decreased jointinflammation when compared to vehicle-treated TNF^(ΔARE/+) mice with aneffect comparable to sc etanercept treated animals (FIG. 16).

ANTI-p40 SIGA EXAMPLES Example 11 Stable Expression of Anti-IL-12/23Secretory Protein Based on Ustekinumab (UKB-SA1) in Lemna a)Construction of Vectors

Synthetic genes were designed for each of the 4 different protein chainsof an anti-IL-12/23 secretory IgA. The amino acid sequence of the heavychain consisted of the rice α-amylase secretion signal (SEQ ID NO:26)joined to the N-terminal amino acid of the variable part of the heavychain of anti-IL12/23 IgG1 antibody Ustekinumab (Stelara®, CAS number815610-63-0, SEQ ID NO:37) which in turn is joined to the N-terminalamino acid of the constant part of a human IgA1 heavy chain (SEQ IDNO:10). The amino acid sequence of the light chain consisted of the riceα-amylase secretion signal (SEQ ID NO:26) joined to the N-terminal aminoacid of the light chain sequence of Ustekinumab (CAS number815610-63-0), which combines an anti-IL-12/23 binding variable part (SEQID NO:38) with a human κ-light chain constant part (SEQ ID NO:14). TheSC-chain consisted of the rice α-amylase secretion signal (SEQ ID NO:26)joined to the N-terminal amino acid of the amino acid sequence of aminoacids 19 to 603 of the human polymeric immunoglobulin receptor disclosedin UniProtKB/Swiss-Prot database entry P01833 (SEQ ID NO:21). TheJ-chain sequence consisted of the rice α-amylase secretion signal (SEQID NO:26) joined to the N-terminal amino acid of the amino acid sequenceof amino acids 23 to 159 of the human sequence disclosed inUniProtKB/Swiss-Prot database entry P01591 (SEQ ID NO:20).

Genes were designed for each of the four components with L. minorpreferred codon usage (63-67% GC content). Tables with suitablepreferred codon use in Lemnaceae can be found in PCT applicationWO2005/035768 and in relevant references contained therein. Restrictionendonuclease sites were added to allow cloning into A. tumefaciensbinary vectors. Design of DNA sequences and vector construction wasperformed by Biolex Therapeutics, Inc., Pittsboro, N.C., USA. DNAsequences were produced by DNA2.0 (Menlo Park, Calif., USA).

The anti-IL-12/23 SIgA (UKB-SA1) was expressed in L. minor bytransfection via an A. tumefaciens binary vector containing DNAsequences encoding all four of the SIgA components: J-chain, SC-chain,H-chain and L-chain. To prepare this vector independent expressioncassettes were created containing promoter, DNA sequences encoding theprotein and terminator for the J-chain (SEQ ID NO:34), SC-chain (SEQ IDNO:36), H-chain (SEQ ID NO:41) and L-chain (SEQ ID NO:42). The H chainwas fused to the modified chimeric octopine and mannopine synthasepromoter with L. gibba 5′ leader from ribulose bis-phosphate carboxylase(RuBisCo) small subunit-1. The L-chain, SC-chain and J-chain genes werefused to high expression Lemnaceae Ubiquitin promoters L. minorpolyubiquitin promoter (LmUbq), L. aequinoctialis polyubiquitin promoter(LaUbq) and Spirodela polyrhiza polyubiquitin promoter (SpUbq),respectively. Sequences of these promoters have been disclosed in PCTapplication WO2007/124186. These expression cassettes were then clonedinto a single A. tumefaciens transformation vector EC2.2 (a modificationof the A. tumefaciens binary vector pBMSP3, which is a derivative ofpBINPLUS. See Ni, M., Cui, D., Einstein, J., Narasimhulu, S., Vergara,C. E., and Gelvin, S. B. Plant J. 7, 661-676, (1995), van EngelenTransgenic Res. 4:288-290 (1995), and Gasdaska et al., Bioprocessing J.,3:50-56 (2003)), with the appropriate restriction sites to create thefinal transformation vector SynA01 (FIG. 18A). This vector alsocontained the gentamycin acetyltransferase-3-I gene (aacC1) whichconfers resistance to gentamycin and allows for selection of transgenicL. minor lines, and was used to produce UKB-SA1 with wild-type(unmodified) N- and O-glycosylation.

SynA01 was used to create additional transformation vectors to generatea glycan optimized version of UKB-SA1, further identified as UKB-SA1g0.A chimeric hairpin RNA was used to silence endogenous L. minor mRNAsencoding α-1,3-fucosyltransferase (Fuct1, GenBank DQ789145) andβ-1,2-xylosyltransferase (Xylt1, GenBank DQ789146). A DNA sequence forthis chimeric RNAi molecule was fused to the high expression SpUbqpromoter and subsequently moved into the SynA01 vector creating thefinal transformation vector SynA02 (FIG. 18B). Also the neomycinphosphotransferase II gene (NPTII) was moved into SynA01 replacing aacC1to produce transformation vector SynA03 (FIG. 6C). This exchange allowsfor kanamycin selection instead of gentamicin selection of transgenicglycan optimized L. minor lines. Further details on procedures forproduction of glycan optimized proteins in Lemnaceae can be found in PCTapplications WO2007/084672, WO2007/084922, WO2007/084926 and in Cox, K.M., Nature Biotechnology 2006, 12: 1591-1597.

b) Transformation of and Expression Using Lemna

Lemna transformation vectors SynA01, SynA02 and SynA03 were transvectedinto A. tumefaciens strain C58Z707 (Hepburn et al., J. Gen. Microbiol.1985, 131: 2961-2969) by electroporation. Agrobacterium colonies wereselected using gentamycin (SynA01 and SynA02) or kanamycin (SynA03) andanalyzed for the presence of the appropriate binary vector using a PCRbased assay. A single colony was selected for each transformation vectorand taken forward into L. minor transformation process (as follows).

Partially dedifferentiated Lemna tissue (L. minor strain 8627) wasincubated with Agrobacterium harboring the expression cassette plasmidby briefly dipping the tissue into the solution. The tissue was thenplaced on co-cultivation plates for two days in continuous light at 25°C. Following co-cultivation, the tissue was transferred to antibioticselection plates and returned to continuous light at 25° C. The tissuewas transferred weekly to fresh antibiotic selection plates. Cefotaximewas included in the antibiotic selection plates to eradicate theAgrobacterium. Gentamicin was included in these plates to select fortransgenic tissue obtained from vectors SynA01 and SynA02 where there isa selectable marker gene included in the transferred genetic cassettewhich confers gentamycin resistance. Kanamycin was included in plates toselect for transgenic tissue from vector SynA03.

UKB-SA1 with unmodified wild-type (WT) glycosylation was obtained bytransfection of L. minor strain 8627 (Biolex Therapeutics Inc.) withvector SynA01. UKB-SA1g0, having G0 glycans lacking fucose and lackingxylose, was obtained by transfecting L. minor strain 8627 with vectorSynA02, or by transfection of the N-glycosylation modified L. minorstrain XF04 (Biolex Inc.) with vector SynA03. Once transformed plantswere regenerated (approximately three months) single plants wereharvested from the antibiotic selection plates and propagated separatelyin liquid growth media, without selection antibiotic, for furtherscreening and characterization. Thus several hundred individualtransgenic plant lines from each construct were generated. Independenttransgenic lines were harvested and clonally propagated in individualharvest jars. For screening of transgenic lines, clonal lines werepreconditioned for 1 week at light levels of 150 to 200 μmol/m²·s invented plant growth vessels containing SH medium (Schenk R. U. et al.,Can. J. Biol. 1972, 50: 199-204) without sucrose. Fifteen to twentypreconditioned fronds were then placed into vented containers containingfresh SH medium, and allowed to grow for two weeks. Tissue samples fromeach line were collected and frozen for analysis.

To determine SIgA expression, frozen tissue samples were homogenized,centrifuged and the supernatant was removed and screened by an ELISAmethod using sheep anti-human IgA secretory chain (AbD Serotec catalog#5111-4804-1:1000 dilution) coated plates to capture the SIgA antibody.The samples were then detected using a goat anti-human kappa light chainHRP conjugated antibody (Sigma catalog #A7164-1:2000 dilution). Thehighest lines from this primary screening were then grown again for twoweeks in small research vessels under the optimal growth conditions, theresulting tissue was harvested and the ELISA was performed to determinethe percent of the total soluble protein that the SIgA antibody isexpressed. The results are summarized in Table 4.

TABLE 4 # of Highest lines Expression Construct Product screened levelSynA01 in UKB-SA1 (WT glycosylation) 262  8.5% TSP L. minor 8627 SynA02in UKB-SA1g0 (G0 N-glycans) 164 15.1% TSP L. minor 8627 SynA03 inUKB-SA1g0 (G0 N-glycans) 452 11.8% TSP L. minor XF04

Example 12 Isolation and Purification of UKB-SA1 and UKB-SA1g0 SecretoryIgA Antibody from Lemna

Biomass from transgenic Lemna expressing UKB-SA1 or UKB-SA1g0, havingvariable regions that are the amino acid sequence of the variableregions (antigen binding regions) of Ustekinumab, was homogenized in 50mM Sodium phosphate, 0.3M Sodium chloride, buffer pH 7.4, at a buffer totissue ratio of 4:1. An acid precipitation step was performed on thecrude extract to remove the enzyme ribulose bis-phosphate carboxylase(RuBisCo) and other plant proteins by adjusting the extract to pH 4.5using 1M Sodium acetate, pH 2.5. The precipitate was removed bycentrifugation of the material at 14,000×g for 30 minutes at 4° C. Thesupernatant was adjusted to pH 7.4 and filtered to 0.22 μm prior to IMACchromatography.

IMAC purification: A Chelating Sepharose FF (GE Healthcare prod. Nr.17-0575-01) column was prepared according to manufacturer instructions(28-4047-39 AC). The column was charged with 3-5 column volumes (cv) of0.1M Copper sulfate. Excess copper was washed with 3-5 cv of doubledistilled water. The column was equilibrated with 3-5 cv of PBS buffer(50 mM Sodium phosphate, 0.15M Sodium chloride, pH 7.4); 3-5 cv of 0.1MSodium acetate, pH 4.0 buffer; 3-5 cv PBS buffer with 0.5M Imidazole;and 3-5 cv PBS buffer.

The supernatant was loaded on the column Approximately 3.8 mg SIgA/mlresin was loaded on columns of up to 350 ml chelating Sepharose.Non-binding material was washed from the column with 10 cv PBS buffer,10 cv of 0.1M Sodium acetate, pH 4.0 buffer, and 10 cv PBS buffer. Theproduct was eluted from the column using 10 cv of PBS buffer containing0.075 M Imidazole (a gradient of 0-0.075 M imidazole was also used butdid not lead to improved results). The fractions containing thesecretory IgA antibodies were pooled.

The column was regenerated by removing copper using a 0.2M EDTA, 0.3Msodium chloride solution, followed by treatment with 0.1N NaOH.

SEC purification: Material obtained by IMAC chromatography was furtherpurified on a Sephacryl 5300 HR column. The column was equilibrated with2 cv of PBS buffer at a flow rate of 0.5 ml/min. The IMAC eluate wasconcentrated 3× using ultra filtration (e.g., using a 5- or 30 kDaregenerated cellulose (hydrosart) membrane) by using, for example, aspin filter (Vivaspin 15R) or a UF cassette (Sartorius 305 144 59 01 E).Typically, a concentration of 5-7 mg/ml and feed volume of 15-20 ml wasused for a 360 ml column. The feed was applied using an AKTA purifier at0.5 ml/min Elution was performed with at least 2 column volumes of PBSbuffer at room temperature and a flow rate of 0.5 ml/min Fractions werecollected and sufficiently pure fractions were pooled.

Results of non-reducing and reducing SDS-PAGE analyses of purifiedmaterial obtained from Lemna transfected with construct SynA01 are shownin FIG. 19 as gels A and B, respectively.

Some batches of material produced by the purification method describedabove were further purified using affinity chromatography usingCaptureSelect human IgA (BAC).

Using an Akta Explorer 10 system a column loaded with 30.9 ml CaptureSelect IgA (BAC) was equilibrated using 3 column volumes (CV) 20 mM TrispH 7.0 buffer at a flow rate of 5 ml/min A solution containing 75.9 mgUKB-SA1 in Tris buffer pH 7.0 was loaded on the column at a flow rate of2.5 ml/min After a wash step with 5 CV of 20 mM Tris buffer pH 7.0 at 5ml/min the product was eluted with 5 CV of 20 mM Tris buffer pH 7.0containing 3.5 M MgCl₂ at 5 ml/min.

The eluate was first dialyzed twice against 5 L 20 mM Tris buffer pH 7.0using snakeskin 10 kDa dialysis tubing for at least 2 hours, followed bydialysis against 5 L PBS puffer pH 7.4 for at least 2 hours. Thesolution was concentrated using a stirred cell (Amicon 8200, Millipore,overhead pressure 30 Psi) with 30 kDa regenerated cellulose membranefilter (Millipore) to a final concentration of approximately 1 mg/ml.The obtained product was further purified using SEC purification asdescribed previously. Glycosylation modified product UKB-SA1g0 waspurified using the same method.

No substantial differences were observed in purification of the twoglycosylation forms, wild-type UKB-SA1 and UKB-SA1g0.

Example 13 Binding of UKB-SA1 and UKB-SA1g0 to IL12

The binding of purified anti-IL-12/23 SIgA, with variable regions takenfrom Ustekinumab and produced in Lemna as in Examples 11 and 12, toIL-12 was determined. The binding of both UKB-SA1 and UKB-SA1g0 productswere determined in comparison to ustekinumab (STELARA®) and colostralSIgA. Plates were coated with IL-12 (Abcam, AB52086) 1 μg/ml. Detectionof bound UKB-SA1/UKB-SA1g0 (secretory IgA) and ustekinumab (IgG1)antibodies was performed using a 1:1500 fold dilution of anti humankappa chain antibody (Abbiotec, cat. no.250987), 100 μl per well, forone hour at RT, washing 3 times, 30 seconds with 200 μl PBS/0.05% Tweenwith shaking, followed by incubation with a 1:1500 fold dilution ofdonkey anti mouse HRP conjugated (Emelca biosciences, MS3001), 100 μlper well, for one hour at RT.

The UKB-SA1, UKB-SA1g0 and ustekinumab antibodies all bound with highaffinity to IL-12 under the conditions of this assay. For UKB-SA1 andUKB-SA1g0 antibodies, binding occurred independent of the type ofglycosylation. Colostral SIgA had minimal to no binding to IL-12.

Example 14 Proteolytic Stability of UKB-SA1 and UKB-SA1g0

The stability of UKB-SA1 and UKB-SA1g0 with antigen binding regionshaving the amino acid sequence of ustekinumab antigen binding regions(i.e., the variable heavy and light chains), produced in Lemna, wasdetermined in simulated intestinal fluid (SIF, 0.05M phosphate buffer pH6.8 containing 10 mg/ml pancreatin). Both the form with wild-type Lemnaglycosylation obtained with vector SynA01, and the G0 glycosylationvariant obtained with vector SynA02 were analysed. Stability wascompared to ustekinumab (IgG1) and to aspecific human colostral SIgAwhich was purified to contain only kappa light chains and α-1 heavychains.

90 μl of a 1 mg/ml solution of the material to be tested was added to810 μl of SIF at 37° C. 50 μl samples were drawn at T=0, 5, 15, 30, 60and 120 min and immediately frozen in liquid nitrogen. Samples wereanalyzed by non-reducing SDS-Page gel electrophoresis. Briefly; 17 μl ofa 0.15M solution of iodoacetamide in LDS sample buffer was added to eachof the frozen samples. Samples were thawed and applied to CriterionTris-HCl gel (12.5%, 18 well, 30 μl comb (Biorad, 345-0015). Afterelectrophoresis gels were treated with Krypton protein stain andanalyzed. Stability of the samples was qualitatively assessed visually.Results are shown in FIG. 20 where gel A is the UKB-SA1 (labeledSynA01-WT in FIG. 20) and gel B is UKB-SA1g0 (labeled SynA02-G0 in FIG.20).

Both UKB-SA1 and UKB-SA1g0 of exhibited a stability that was comparableto natural human colostral SIgA. The IgG1 antibody ustekinumab degradedunder these conditions at such a high rate that detection at T=0 was notpossible.

Example 15A Inhibition of IL-12/23 in Dendritic Cells

To demonstrate inhibition of endogenous produced p40/IL12/IL23 byUKB-SA1 or UKB-SA1g0 versus ustekinumab (IgG1) or non-specific SIgA(polyclonal colostral SIgA), in vitro experiments were performed withdendritic cells and T cells. Monocytic cells were isolated from humandonor blood and stimulated with lipopolysccharide (LPS) and the cellswere differentiated into mature dendritic cells. The stimulation withLPS and resulting differentiation was performed in the presence of aSIgA antibody of the invention, Ustekinumab, polyclonal colostral SIgA,or without any added antibody (i.e., LPS only).

In more detail, the experiments were performed as follows. Humandendritic cells (DCs) were obtained from isolated monocytes from buffycoats. The adherent monocytes were cultured in synthetic X-VIVO 15medium (Lonza, Cat. No. BE04-418Q) supplemented with 2% of AB humanserum (Sigma, Cat. No. H4522), 450 U/ml GM-CSF (Miltenyi Biotec, Cat.No. 130-093-867) and 300 U/ml IL-4 (Miltenyi Biotec, Cat. No.130-093-924) as growth and differentiation factors, respectively, for 6days to obtain immature DCs. After one week, immature DCs (5×10³) wereactivated for 24 hours with 1 μg/ml LPS (Invivogen, Cat. No. tlrl-pelps)in the presence of different concentrations UKB-SA1, UKB-SA1g0, orcontrol antibodies to induce DC maturation. IL12/IL23 concentration inculture medium was measured by ELISA according to the manufactures'instructions (BD OptEIA, Cat. No. 555171).

The results are represented in FIG. 21A where open circle is UKB-SA1;closed circle is UKB-SA1g0; Diamond is Colostral SIgA; and Square isustekinumab. Data are means (+/−SEM) of 3 experiments. Both UKB-SA1 andUKB-SA1g0 inhibited endogenous produced p40/IL12/IL23 by dendriticcells. Both UKB-SA1 and UKB-SA1g0 had a similar efficacy and potency asUstekinumab in this regard.

Example 15B Inhibition of IL-12/23 in Dendritic Cells

Similar to above, inhibition of IL-12/23 in dendritic cells was measuredbut at a fixed concentration of the antibody. Human dendritic cells(DCs) were obtained from isolated monocytes from buffy coats. Theadherent monocytes were cultured in synthetic X-VIVO 15 medium (Lonza,Cat. No. BE04-418Q) supplemented with 2% of AB human serum (Sigma, Cat.No. H4522), 450 U/ml GM-CSF (130-093-867, Miltenyi Biotec) and 300 U/mlIL-4 (Miltenyi Biotec, Cat. No. 130-093-924) as growth anddifferentiation factors, respectively, for 6 days to obtain immatureDCs. After one week, immature DCs (5×10³) were activated for 24 hourswith 1 μg/ml LPS (Invivogen, Cat. No. thl-pelps) in the presence of 10μg/ml UKB-SA1, UKB-SA1g0, or control antibodies to induce DC maturation.IL12/IL23 concentration in culture medium was measured by ELISAaccording to the manufactures' instructions (BD OptEIA, Cat. No.555171).

The results are represented in FIG. 21B. Both UKB-SA1 (labeled SynA01 inFIG. 21B) and UKB-SA1g0 (labeled SynA02 in FIG. 21B) inhibitedendogenous produced p40/IL12/IL23 by dendritic cells to the same extentas ustekinumab.

Example 15C Inhibition of IL-12/23 in Dendritic Cells and T Cells

To determine if other “intrinsic” properties were affected by theantibodies, dendritic cells were co-cultured with T-cells. The crosstalkbetween these cells, which might result in a change of phenotype of thedendritic cells, or cytokine secretion profile of the T-cells wasexamined. DCs were stimulated with LPS for 6 h; afterwards DCs werewashed twice in PBS and in the presence of 10 μg/ml of UKB-SA1,UKB-SA1g0 or control antibodies cultured with allogeneic T cells. Theallo-response was tested in a mixed lymphocyte reaction; allogeneic Tcells (10⁵ cells) were co-cultured in triplicate with differentlytreated DCs (5*10³) in a 96-well round bottom plate, in 200 μl of medium(X-VIVO 15+2% HS). For T helper polarisation T lymphocytes supernatantswere collected at day 4 and T helper specific cytokine IFNγ was analysedby ELISA (BD OptEIA, Cat. No. 555142), according to the manufacturers'instructions.

The results are represented in FIG. 21C. UKB-SA1 (labeled SynA01 in FIG.21C) and UKB-SA1g0 (labeled SynA02 in FIG. 21C) blocked the IFNγsecretion by co-cultured DCs and T-cells to the same extent asUstekinumab, indicating an inhibition of proinflammatory T helper-1cells.

Example 15D Inhibition of IL-12/23 in Dendritic Cells and T Cells

Similar to above, the antibodies were applied in fixed concentrations tothe DC and T cells. DCs were stimulated with LPS for 6 h; afterwards DCswere washed twice in PBS and in the presence of 10 μg/ml of UKB-SA1,UKB-SA1g0 or control antibodies cultured with allogeneic T cells. Theallo-response was tested in a mixed lymphocyte reaction; allogeneic Tcells (10⁵ cells) were co-cultured in triplicate with differentlytreated DCs (5*10³) in a 96-well round bottom plate, in 200 μl of medium(X-VIVO 15+2% HS). For T helper polarisation T lymphocytes supernatantswere collected at day 4 and T helper specific cytokine IL-10 wasanalysed by ELISA (eBioscience Cat. No. 88-7106-88), according to themanufacturers' instructions.

The results are represented in FIG. 21D. UKB-SA1 (labeled SynA01 in FIG.21D) and UKB-SA1g0 (labeled SynA02 in FIG. 21D) had no effect on theIL-10 secretion by co-cultured DCs and T-cells.

Example 16 Pharmacokinetics of UKB-SA1g0

Serum concentrations of UKB-SA1g0 after oral and intravenousadministration were determined UKB-SA1g0 was administered to C57Bl/6mice (200 μg for intravenous, 400 μg oral) and blood samples were takenby orbital bleeding at different time points. UKB-SA1g0 was measured byan in-house constructed ELISA. A goat anti-human kappa light chainantibody (SouthernBiotech, Cat. No. SBA 2060-02) was used for coating ofthe plates, and a goat anti-human IgA antibody (SouthernBiotech, Cat.No. SBA 2050-05) for detection. The minimum detection limit of the ELISAis 5 ng/ml.

The results are represented in FIG. 22 wherein circle shows oraladministration and square shows intravenous administration. Dotted lineat 5 ng/ml depicts the detection limited of the ELISA used forquantification. Data are mean+/−SD of 2 representative mice. UKB-SA1g0(labeled SynA02 in FIG. 22) is rapidly cleared from circulation afterintravenous injection and very little (if any) exposure was measuredafter oral administration.

Example 17 In Vivo Administration of SIgA and IgGAnti-IL12/23Pharmacokinetics

Using in vivo imaging of fluorescent labeled antibodies, images ofthoracic and abdominal region of mice can reveal distribution of theantibody over time. UKB-SA1 was conjugated with either Alexa Flour 647(Molecular probes), or DyLight 755 (ThermoFisher) according manualinstructions. The dye can be detected as a fluorescent signal with theMaestro in vivo imaging system (Cambridge Research & Instrumentation).Fluorescent conjugated antibodies (100 μg/mice) were administered orallyor intraperitoneally. Mice were anaesthetized with Xylamine/Ketaminebefore examination with the Maestro system in order to immobilizeanimals during picture taking procedure. Then mice were analyzed andpictures of the distribution of the fluorescence conjugated UKB-SA1 weretaken at different time points indicated at the experimentally setup.After that background fluorescence were subtracted by software.

The results are represented in FIG. 23 wherein the first three columns(^(a)) correspond to the DyLight 775 conjugate and the fourth column(^(b)) is the UKB-SA1 Alexa Flour 647 conjugate. First hours after oraladministration fluorescent conjugated antibodies UKB-SA1 (labeled SynA01in FIG. 23) and IgG1 Ustekinumab (labelled IgG in FIG. 23) are presentin thoracic area of mice, probably the stomach, whereas after 24 hours aclear signal of UKB-SA1, but not IgG1, is present in the abdominalregion (intestine) of the mice. Intraperitoneal administration resultsin a diffuse thoracic and abdominal signal after 24 hours.

Example 18 Distribution In Vivo after Oral Administration

Immunohistochemistry on cryo-sections of mice distal colon, 48 hoursafter oral administration of antibody were assessed. Antibodies (100μg/mouse) were administered orally and after 48 hours animals weresacrificed and intestinal tract isolated. Cry-section were stained withanti-SC-chain antibody (Santa Cruz, Cat. No. sc20656) for detection ofantibodies containing a human secretory chain. This antibody does notcross-react with mouse secretory chain. As secondary antibodygoat-anti-rabbit conjugated with Cy3-red antibody (JacksonImmunoresearch, Cat. No. 111-166-045) was used.

The results are represented in FIG. 24 where the left side is UKB-SA1(labeled SynA01 in FIG. 24) and the right side is IgG1 Ustekinumab(labeled IgG in FIG. 24). UKB-SA1 is present in cells of the sub mucosallayer of the distal colon as indicated by the bright spots in thevicinity of the white arrows. No IgG1 is indicated as being present inthe distal colon cells.

Example 19 Efficacy of UKB-SA1 and UKB-SA1g0 in In-Vivo Animal Models ofIBD

Heparinized blood was obtained from healthy donors (buffy coats). PBMC'swere isolated from buffy coats by Ficoll-Paque 1.077 g/ml (PAALaboratories GmbH) density centrifugation and cells were washed with1×PBS before reconstitution. NOD.CB17-Prkdcscid/J γc−/− (NOD-SCID-IL2receptor deficient) mice 4 to 8 weeks of age were reconstituted with30×10⁶ human cells. Three weeks after reconstitution animals werepre-treated orally 5 times a week (not in weekend) with PBS control,UKB-SA1 (100 μg/mouse), UKB-SA1g0 (100 μg/mouse), or 3 times per weeksubcutaneous with Ustekinumab (100 μg/mouse). Start of pre-treatment ist=0. At day t=7 reconstituted mice were treated with 2% dextran sulfatesodium (DSS) in drinking water to induce inflammation symptoms inintestine. First signs of inflammation can be seen at day 10-12,together with declined body weight. With mini-endoscopic system ananalysis of the status of disease was performed. Briefly, amini-endoscope (1.9 mm outer diameter) was introduced via the anus andthe colon was carefully inflated with an air pump. Endoscopic picturesobtained are of high quality and allow the monitoring and grading ofinflammation. Thereafter, endoscopic scoring of five parameters from 0-3(1; translucent structure, 2; granularity, 3; fibrin, 4; vascularity,and 5; stool) resulting in the overall score from 0 (no change) to 15(severe colitis) was performed. Typically, after one week exposure toDSS the symptoms are so strong that mice need a resting period torecover. At day t=16 the experiment was terminated and clinical scoreanalyzed.

The results are depicted in FIGS. 25A and B. Oral treatment with UKB-SA1(labeled SynA01 in FIGS. 25A and 25B) and UKB-SA1g0 (labeled SynA02 inFIGS. 25A and 25B), as well as subcutaneous treatment with Ustekinumab,significantly inhibited DSS-induced inflammatory bowel disease inhumanized-scid mice. Statistical analysis: ANOVA followed by Dunnett'sMultiple Comparison test, all groups were compared to PBS controlgroup. * p<0.05, *** p<0.001, n=7 mice per group. FIG. 25B depicts arepresentative mini-endoscopic picture of the colitis score at day t-15.

Example 20 Efficacy of UKB-SA1 and UKB-SA1g0 in Human Psoriatic SkinBiopsies

The activity of SIgA anti-p40 on gene expression was evaluated for alimited number of selected genes in skin explants of psoriatic lesionsin vitro. Psoriatic cutaneous lesions can be maintained in culture up to8 days without significant changes in their psoriatic phenotype. Twopunch biopsies of 4 mm were obtained from 7 psoriasis patients(volunteers). Time of evolution of the lesion varied between 1 and 12months. The two biopsies from each patient were cut in half making four2 mm biopsies. From one punch, one 2 mm biopsy was tested with vehicleand the other with UKB-SA1. From the other punch, one 2 mm biopsy astested with UKB-SA1g0 and the other with ustekinumab. UKB-SA1 andUKB-SA1g0 were tested at equimolar concentrations of 14 μg/ml andUstekinumab was tested at 10 μg/ml. The biopsies, medium, and antibodyas appropriate were combined in a 24 well plate (800 μl) for 8 days withthe medium changed every 2-3 days. After 8 days of culture, the skinbiopsies were frozen in liquid nitrogen and then evaluated. Genes wereevaluated by RTPCR: Beta-defensin 4, Keratin 16, Interleukin-10, andGAPDH (house-keeping gene). Keratin 16 and β-Defensin 4 are proteinsexpressed in epithelial tissue and related to skin diseases.Down-regulation of these markers anticipate clinical efficacy ofanti-inflammatory treatment in the clinic. Gene expression in treatedbiopsies was normalized to house-keeping gene GAPDH according to theformula 1,8e (Ct GADPH-Ct gene of interest)×10000 (Ct=cycle thresholdtime). After normalization, gene expression of gene of interest wascompared to gene expression in vehicle-treated biopsies.

The results are represented in the table below:

Time of evolution of the Patient No. lesion (months) SynA01 SynA02Ustekinumab 2 12 K16 ++ K16 − K16 = BF +++ BF = BF +++ IL10 −−− IL10 −−IL10 ++ 5 12 K16 = K16 + K16 = BF +++ BF +++ BF ++ IL10 − IL10 − IL10 =4 6 K16 + K16 +++ K16 +++ BF +++ BF + BF +++ IL10 + IL10 + IL10 = 3 3K16 = K16 + K16 = BF = BF −− BF −− IL10 ++ IL10 + IL10 = 7 2 K16 +++ K16+++ K16 +++ BF −−− BF −−− BF −−− IL10 −−− IL10 −−− IL10 −−− 6 1 K16 −−−K16 ++ K16 −− BF −− BF +++ BF −−− IL10 +++ IL10 +++ IL10 − 1 1 K16 −−K16 − K16 − BF −− BF +++ BF − IL10 ++ IL10 +++ IL10 − Legend:Beta-defensin 4 (BF4), Keratin 16 (K16), Interleukin 10 (IL-10) Noregulation (0-10% difference in gene expression) is “=”; Up-regulated(11-25% difference in gene expression) is +; 26-50% is ++; and >51% is+++. Down-regulated (11-25% difference in gene expression) is −; 26-50%is −−; and >51% is −−−.

Time of evolution of the lesion appears to be relevant for the activityof antip40 antibodies as UKB-SA1, UKB-SA1g0, and ustekinumab haveactivities in patients, especially those with less than 6 months ofevolution of the lesion. Gene expression of Keratin 16 and Beta defensin4 (two) were reduced in young lesions (1-3 month) by SynA01, SynA02 andustekinumab, indicating reduced epithelial activity and a treatmenteffect.

In these young lesions, Interleukin-10 expression was increased only byUKB-SA1 and UKB-SA1g0, but not by ustekinumab, indicating an additionalanti-inflammatory cytokine response of SIgA's compared to an IgGantibody.

Each of the patents, patent applications, and journal articles mentionedabove are incorporated herein by reference in their entirety. Theinvention having been described it will be obvious that the same may bevaried in many ways and all such modifications are contemplated as beingwithin the scope of the invention as defined by the claims appendedhereto.

1. A monoclonal secretory IgA antibody, which binds to and neutralizes ahuman proinflammatory cytokine.
 2. The antibody according to claim 1,wherein said antibody is a chimeric antibody.
 3. The antibody accordingto claim 1, wherein said antibody is a humanized antibody or a humanantibody.
 4. The antibody according to any of claims 1-3, wherein saidantibody comprises a human secretory chain and a human J-chain.
 5. Theantibody according to claim 4, wherein said human secretory chain hasthe sequence of SEQ ID NO:21 and said human J-chain has the sequence ofSEQ ID NO:20.
 6. The antibody according to any of claims 1-5, whereinsaid antibody is a human secretory IgA1 antibody.
 7. The antibodyaccording to any of claims 1-6, wherein said human proinflammatorycytokine is selected from the group consisting of TNFα (soluble),interferon gamma, interferon alpha, GM-CSF, CXCL10/IP-10, IL-1β, IL-1α,IL-4, IL-5, IL-6, IL-12; IL-13, IL-17A, IL-18, IL-20, IL-22, and IL-23.8. The antibody according to any of claims 1-6, wherein said antibodycomprises CDR sequences that are identical to the CDR sequences of anantibody selected from the group consisting of adalimumab, infliximab,golimumab, certolizumab pegol, ozoralizumab, sifalimumab, canakinumab,gevokizumab, pascolizumab, reslizumab, mepolizumab, sirukumab,olokizumab, ustekinumab, briakinumab, tralokinumab, anrukinzumab,lebrikizumab, secukinumab, ixekizumab, and fezakinumab.
 9. A secretoryIgA composition, comprising a plurality of antibodies according to anyof claims 1-8, wherein substantially all N-glycans in said plurality ofantibodies lack fucose and xylose residues.
 10. A secretory IgAcomposition, comprising a plurality of antibodies according to any ofclaims 1-8, wherein said plurality of antibodies contains at least about30% G0 glycans (preferably G0 glycans lacking Fuc and Xyl residues)relative to the total amount of N-glycans in the population.
 11. Asecretory IgA composition, comprising a plurality of antibodiesaccording to any of claims 1-8, wherein said plurality of antibodiescontains at least about 25% high-mannose glycans (e.g., Man5, Man6,Man7, Man8, and/or Man9 glycans) relative to the total amount ofN-glycans in the population.
 12. The composition according to any ofclaims 10-11, wherein G0 glycans (preferably G0 glycans lacking Fuc andXyl residues) and high-mannose glycans (e.g., Man5, Man6, Man7, Man8,and/or Man9 glycans) together are the majority of glycans present insaid plurality of antibodies.
 13. The composition according to claim 12,wherein said G0 glycans and said high-mannose glycans together are atleast 70% of the total amount of N-glycans in said plurality ofantibodies.
 14. A pharmaceutical composition, comprising the antibodyaccording to any of claims 1-8, or the composition according to any ofclaims 9-13, and at least one pharmaceutically acceptable excipient. 15.The pharmaceutical composition according to claim 14, wherein saidcomposition is adapted for oral administration.
 16. The secretory IgAantibody according to any of claims 1-8, or the composition of accordingto any of claims 9-15, for use as a medicament preferably for thetreatment of an inflammatory disease in a human.
 17. A method fortreating an inflammatory disease in a human, which comprisesadministering an anti-inflammatory effective amount of the antibodyaccording to any of claims 1-8, or the composition according to any ofclaims 9-15, to a human in need thereof.
 18. The method according toclaim 17, wherein said administering comprises orally administering saidantibody to said human.
 19. The method according to any of claims 17-18,wherein said inflammatory disease is selected from the group consistingof rheumatoid arthritis, inflammatory bowel disease (including Crohn'sdisease and ulcerative colitis), psoriasis, psoriatic arthritis,ankylosing spondylitis, juvenile idiopathic arthritis, uveitis, asthma,Alzheimer's disease, multiple sclerosis, type II diabetes, systemicsclerosis, lupus nephritis, and allergic rhinitis.
 20. A monoclonalsecretory IgA antibody, which binds to and blocks a humanproinflammatory cytokine receptor selected from the group consisting ofIL-1R, IL-2R, IL-4R, IL-5R, IL-6R, and IL-17R.